Method for producing 3-hydroxypropionic acid and other products

ABSTRACT

This invention relates to metabolically engineered microorganism strains, such as bacterial strains, in which there is an increased utilization of malonyl-CoA for production of a chemical product, which includes 3-hydroxypropionic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityto from U.S. patent application Ser. No. 14/575,927, filed on Dec. 18,2014, which is a continuation of and claims the benefit of priority toU.S. patent application Ser. No. 13/916,534, filed on Jun. 12, 2013,which is a continuation of and the claims the benefit of priority toU.S. patent application Ser. No. 13/498,468, filed on Feb. 12, 2013, nowissued as U.S. Pat. No. 8,883,464, which claims the benefit of priorityto National Stage Application No. PCT/US2010/050436, filed on Sep. 27,2010, which claims the benefit of priority to U.S. ProvisionalApplication 61/246,141, filed Sep. 27, 2009, U.S. ProvisionalApplication 61/298,844, filed Jan. 27, 2010, and U.S. ProvisionalApplication 61/321,480, filed Apr. 6, 2010. The entire contents of eachapplication are hereby incorporated by reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Contract No.DE-AR0000088 awarded by the department of Energy. The Government hascertain rights in this invention.

FIELD OF TILE INVENTION

This invention relates to metabolically engineered microorganisms, suchas bacterial strains, in which there is an increased utilization ofmalonyl-CoA for production of a chemical product, which may include thechemical 3-hydroxypropionic acid (3-HP) and products made from 3-HP. Themetabolically engineered microorganisms may be adapted to exhibitincreased tolerance to 3-HP. Also, genetic modifications may be made toprovide one or more 3-HP biosynthesis pathways such as in microorganismscomprising one or more genetic modifications of a complex identified asthe 3-HP toleragenic complex.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 27, 2016, isnamed 1583297.TXT and is 2,232,320 bytes in size.

BACKGROUND OF THE INVENTION

With increasing acceptance that petroleum hydrocarbon supplies aredecreasing and their costs are ultimately increasing, interest hasincreased for developing and improving industrial microbial systems forproduction of chemicals and fuels. Such industrial microbial systemscould completely or partially replace the use of petroleum hydrocarbonsfor production of certain chemicals.

Numerous chemicals are produced through such means, ranging fromantibiotic and anti-malarial pharmaceutical products to fine chemicalsto fuels such as ethanol. Commercial objectives for microbialfermentation include the increase of titer, production rate, and yieldof a target chemical product. When the overall specific productivity ina fermentation event is elevated, this may positively affect yield inaddition to production rate and other economic factors, such as capitalcosts.

One candidate chemical for such production is 3-hydroxypropionic acid(“3-HP”, CAS No. 503-66-2), which may be converted to a number of basicbuilding blocks for polymers used in a wide range of industrial andconsumer products. Unfortunately, previous efforts to microbiallysynthesize 3-HP to achieve commercially viable titers have revealed thatthe microbes being used were inhibited by concentrations of 3-HP farbelow a determined commercially viable titer.

In spite of strong interest to improve microbial fermentation economicsby improving yield and/or productivity for certain chemical products,there remains a need to increase net conversion in a fermentativemicroorganism cell to desired target chemical products employingcommercially viable fermentation methods. More particularly, amongproblems remaining, to be solved are how to improve specificproductivity and volumetric productivity, such as to economicallyimportant levels, in modified microorganisms that are adapted to producea chemical product having malonyl-CoA as a substrate in the microbialproduction pathway of that chemical product, such as 3-hydroxypropionicacid (3-HP).

SUMMARY OF THE INVENTION

According to one embodiment, the invention is directed to a method forproducing an acrylic acid-based consumer product, said method comprisingi) combining a carbon source and a microorganism cell culture to produce3-hydroxypropionic acid, wherein a) said cell culture comprises aninhibitor of fatty acid synthase or said microorganism is geneticallymodified for reduced enzymatic activity in the organism's fatty acidsynthase pathway; or b) wherein said microorganism is geneticallymodified for increased enzymatic activity in the organism's malonyl-CoAreductase (mcr) pathway by introduction of a heterologous nucleic acidsequence coding for a polypeptide having mono-functional malonyl-CoAreductase activity; or c) said 3-hydroxypropionic acid is produced at aspecific productivity of greater than 0.05 grams per gram ofmicroorganism cell on a dry weight basis per hour or at a volumetricproductivity of greater than 0.50 grams per liter per hour; ii)converting the 3-hydroxypropionic acid to acrylic acid; and iii)processing the acrylic acid into a consumer product. In various aspects,the carbon source has a ratio of carbon-14 to carbon-12 of about1.0×10⁻¹⁴ or greater.

The carbon source according to the invention may be predominantlyglucose, sucrose, fructose dextrose, lactose, or a combination thereof.Alternatively, the carbon source is glycerol.

In certain embodiments, the cell culture comprises an inhibitor of fattyacid synthase or said microorganism is genetically modified for reducedenzymatic activity in the organism's fatty acid synthase pathway. Forexample, the inhibitor of a fatty acid synthase may be selected from thegroup consisting of thiolactomycin, triclosan, cerulenin,thienodiazaborine, isoniazid, and analogs thereof.

The microorganism of the invention may be genetically modified forincreased enzymatic activity organism's malonyl-CoA reductase (mcr)pathway by introduction of a heterologous nucleic acid sequence codingfor a polypeptide having mono-functional malonyl-CoA reductase activity.In various embodiments, the mono-functional malonyl-CoA reductase isNADPH-independent.

In various embodiments, the 3-hydroxypropionic acid is producedaccording to the invention at a specific productivity of greater than0.05 grams per gram of microorganism cell on a dry weight basis per houror at a volumetric productivity of greater than 0.05 grams per liter perhour.

Included within the invention are embodiments where the cell culturecomprises a genetically modified microorganism. The genetically modifiedmicroorganism can be modified for a trait selected from reducedenzymatic activity in the organism's fatty acid synthase pathway,increased enzymatic activity in the organism's malonyl-CoA reductasepathway, increased tolerance to 3-hydroxypropionic acid, increasedenzymatic activity in the organism's NADPH-dependent transhydrogenasepathway, increased intracellular bicarbonate levels, increased enzymaticactivity in the organism's acetyl-CoA carboxylase pathway, andcombinations thereof. For example, the genetically modifiedmicroorganism can be modified for reduced enzymatic activity in theorganism's fatty acid synthase pathway. Alternatively, the reducedenzymatic activity is a reduction in enzymatic activity in an enzymeselected from the group consisting of beta-ketoacyl-ACP reductase,3-hydroxyacyl-CoA dehydratase, enoyl-ACP reductase, and thioesterase, invarious aspects, the reduced enzymatic activity in the organism's fattyacid synthase pathway occurs via introduction of a heterologous nucleicacid sequence coding for an inducible promoter operably linked to asequence coding for a enzyme in the fatty acid synthase pathway orhomolog thereof, or a heterologous nucleic acid sequence coding for anenzyme in the fatty acid synthase pathway or homolog thereof withreduced activity. In various aspects, the enzyme in the fatty acidsynthase pathway or homolog thereof is a polypeptide withtemperature-sensitive beta-ketoacyl-ACP or temperature-sensitiveenoyl-ACP reductase activity. Variously, the genetically modifiedmicroorganism is modified for increased enzymatic activity in theorganism's malonyl-CoA reductase pathway.

In certain embodiments, the increase in enzymatic activity in themalonyl-CoA reductase (mcr) pathway occurs by introduction of aheterologous nucleic acid sequence coding for a polypeptide havingbi-functional malonyl-CoA reductase enzymatic activity ormono-functional malonyl-CoA reductase activity. The heterologous nucleicacid sequence may be selected from a sequence having at least 70%homology with a sequence selected from SEQ ID NO. 780-789.

In various embodiments, the genetically modified microorganism ismodified for increased tolerance to 3-hydroxypropionic acid. Theincrease in tolerance to 3-hydroxypropionic acid may occur in one ormore components of the 3-HP toleragenic complex (3HPTGC) complex, orwherein said increase in tolerance to 3-hydroxypropionic acid resultsfrom providing at least one genetic modification of each of Group A andGroup B of the 3HPTGC. The one or more components may be selected fromCynS, CynT, AroG, SpeD, SpeE, SpeF, ThrA, Asd, CysM, IroK, IlvA, andhomologs thereof. In various embodiments, the modification is adisruption of one or more 3HPTGC repressor genes. The repressor genesmay be selected front tyrR, trpR, metJ, purR, lysR, nrdR, and homologsthereof.

Increased enzymatic activity in the organism's NADPH-dependenttranshydrogenase pathway may occur by introduction of a heterologousnucleic acid sequence coding for a polypeptide having at least 70%homology with a sequence selected from SEQ ID NO. 776 or 778. In variousembodiments, the increased intracellular bicarbonate levels occurs byintroduction of a heterologous nucleic acid sequence coding for apolypeptide having cyanase and/or carbonic anhydrase activity.Heterologous nucleic acid sequence may be selected from a sequencehaving at least 70% homology with a sequence selected from SEQ ID NO.337.

In various embodiments, an increased enzymatic activity in theorganism's acetyl-CoA carboxylase pathway occurs by introduction of aheterologous nucleic acid sequence coding for a polypeptide having atleast 70% homology with a sequence selected from SEQ ID NO. 768-775.

The genetically modified bacteria may be further modified to decreaseactivity of, lactate dehydrogenase, phophate acetyltransferase, pyruvateoxidase, or pyruvate-formate lyase, and combinations thereof.

The method according to the invention may further comprise separatingand/or purifying 3-hydroxypropionic acid from said cell culture byextraction of 3-hydroxypropionic acid from said culture in the presenceof a tertiary amine. Variously, 3-hydroxypropionic acid is produced at aspecific productivity of greater than 0.05 grams per gram ofmicroorganism cell on a dry weight basis per hour or at a volumetricproductivity of greater than 0.50 grams per liter per hour.

The method of the invention may include production of a consumerproduct, such as diapers, carpet, paint, adhesives, and acrylic glass.The invention includes biologically-produced 3-hydroxypropionic acid,where the 3-hydroxypropionic acid is produced according to the method ofthe invention. Such 3-hydroxypropionic acid may be essentially free ofchemical catalyst, including a molybdenum and/or vanadium basedcatalyst. The 3-hydroxypropionic acid is produced according to themethod of the invention may have a ratio of carbon-14 to carbon-12 ofabout 1.0×10⁻¹⁴ or greater. In various aspects, the 3-hydroxypropionicacid contains less than about 10% carbon derived from petroleum. Inaddition, 3-hydroxypropionic acid according to the invention may containa residual amount of organic material related to its method ofproduction. In various embodiments, the 3-hydroxypropionic acid containsa residual amount of organic material in an amount between 1 and 1,000parts per million of the 3-hydroxypropionic acid.

Acrylic acid and a polymer produced from acrylic acid, where such areproduced according to the method of the invention, are also includedwithin the invention. Products, including commercial and consumerproducts, obtained from the polymers are also encompassed. For example,diapers, carpet, paint, adhesives, and acrylic glass are encompassed.

In addition, the invention encompasses a system for bioproduction ofacrylic acid, said system comprising: a tank for saccharification ofbiomass; a line for passing the product of saccharification to afermentation tank optionally via a pre-fermentation tank;a fermentationtank suitable for microorganism cell culture; a line for dischargingcontents from the fermentation tank to an extraction and/or separationvessel; an extraction and/or separation vessel suitable for removal of3-hydroxypropionic acid from cell culture waste; a line for transferring3-hydroxypropionic acid to a dehydration vessel; and a dehydrationvessel suitable for conversion of 3-hydroxypropionic acid to acrylicacid. In various embodiments, the system further comprises one or morepre-fermentation tanks, distillation columns, centrifuge vessels, backextraction columns, mixing vessels, or combinations thereof. In variousembodiments, the system has a minimum production capacity of at least 1ton acrylic acid per year.

Within the scope of the invention are genetically modifiedmicroorganism, wherein the microorganism is capable of producing3-hydroxypropionate at a specific rate selected from the rates ofgreater than 0.05 g/gDCW-hr, 0.08 g/gDCW-hr, greater than 0.1 g/gDCW-hr,greater than 0.13 g/gDCW-hr, greater than 0.15 g/gDCW-hr, greater, than0.175 g/gDCW-hr, greater than 0.2 g/gDCW-hr, greater than 0.25g/gDCW-hr, greater than 0.3 g/gDCW-hr, greater than 0.35 g/gDCW-hr,greater than 0.4 g/gDCW-hr, greater than 0.45 g/gDCW-hr, or greater than0.5 g/gDCW-hr.

The genetically modified microorganism may comprise geneticmodifications to increase malonyl-coA reductase activity and acetyl-coAcarboxylase activity, and genetic modifications to reduce enoyl-ACPreductase activity, lactate dehydrogenase activity and acetate kinaseactivity. Variously, the microorganism comprises genetic modificationsto increase malonyl-coA reductase activity and acetyl-coA carboxylaseactivity, and genetic modifications to reduce enoyl-ACP reductaseactivity, lactate dehydrogenase activity and acetylphosphate transferaseactivity. In addition, the microorganism may comprise geneticmodifications to increase malonyl-coA reductase activity and acetyl-coAcarboxylase activity, and genetic modifications to reduce enoyl-ACPreductase activity, lactate dehydrogenase activity, acetate kinaseactivity and acetylphosphate transferase activity. In various aspects,the microorganism comprises genetic modifications to increasemalonyl-coA reductase activity and acetyl-coA carboxylase activity, andgenetic modifications to reduce enoyl-ACP reductase activity, lactatedehydrogenase activity and pyruvate formate lyase activity. In variousembodiments, the microorganism comprises genetic modifications toincrease malonyl-coA reductase activity and acetyl-coA carboxylaseactivity, and genetic modifications to reduce enoyl-ACP reductaseactivity, lactate dehydrogenase activity and pyruvate oxidase activity.Also included are microorganisms comprising genetic modifications toincrease malonyl-coA reductase activity and acetyl-coA carboxylaseactivity, and genetic modifications to reduce enoyl-ACP reductaseactivity, lactate dehydrogenase activity and methylglyoxal synthaseactivity. In addition, microorganisms according to the invention maycomprise genetic modifications to increase malonyl-coA reductaseactivity and acetyl-coA carboxylase activity, and genetic modificationsto increase β-ketoacyl-ACP synthase activity, and decrease lactatedehydrogenase activity and methylglyoxal synthase activity, and/or themicroorganism may comprise genetic modifications to increase malonyl-coAreductase activity and acetyl-coA carboxylase activity, and geneticmodifications to reduce enoyl-ACP reductase activity, guanosine3′-diphosphate 5′-triphosphate synthase activity, and guanosine3′-diphosphate 5′-diphosphate synthase activity. Also, in somemicroorganisms enoyl-CoA reductase, is reduced instead of or in additionto doing such for enoyl-ACP reductase activity.

In various embodiments, a further genetic modification has been madethat increases NADH/NADPH transhydrogenase activity. For example, thetranshydrogenase activity may be soluble, may be membrane bound, mayhave a further genetic modification that has been made that increasescyanase activity, may include a further genetic modification thatincreases carbonic anhydrase activity, and/or may include a furthergenetic modification that increases pyruvate dehydrogenase activity.

In various embodiments, a further genetic modification has been madethat decreases guanosine 3′-diphosphate 5′-triphosphate synthaseactivity, and guanosine 3′-diphosphate 5′-diphosphate synthase activity.Also included is when a genetic modification has been made thatincreases the NADH/NAD+ ratio in an aerated environment. Further, agenetic modification may be made that decreases β-ketoacyl ACP synthaseactivity, decreases 3-hydroxypropionate reductase activity, decreasesNAD+ dependant 3-hydroxypropionate dehydrogenase activity, decreasesNAD+ dependant 3-hydroxypropionate dehydrogenase activity, increasestolerance to 3-hydroxypropionic acid, increases activity of any enzymein the 3-HP toleragenic complex, increases pyruvate dehydrogenaseactivity, increases cyanase activity, increases carbonic anhydraseactivity, increases aspartate kinase activity, increases threoninedehydratase activity, increases 2-dehydro-3-deoxyphosphoheptonateaidolase activity, increases cysteine synthase activity, increasesribose-phosphate diphosphokinase activity, increasesribonucleoside-diphosphate reductase activity, increases L-cysteinedesulfhydrase activity, increases lysine decarboxylase activity,increases homocysteine transmethylase activity, increases dihydrofolatereductase activity, increases N-acetylglutamylphosphate reductaseactivity, increases acetylglutamate kinase activity, increasesargininosuccinate lyase activity, increases acetylornithine deacetylaseactivity, increases chorismate mutase activity, increases prephenatedehydratase activity, increases prephenate dehydrogenase activity,increases 2-dehydro-3-deoxyphosphoheptonate aldolase activity, and/orincreases D-3-phosphoglycerate dehydrogenase activity.

In various embodiments, the invention includes a culture systemcomprising a carbon source in an aqueous medium and a geneticallymodified microorganism, wherein said genetically modified organism ispresent in an amount selected from greater than 0.05 gDCW/L, 0.1 gDCW/L,greater than 1 gDCW/L, greater than 5 gDCW/L, greater than 10 gDCW/L,greater than 15 gDCW/L or greater than 20 gDCW/L, such as when thevolume of the aqueous medium is selected from greater than 5 mL, greaterthan 100 mL, greater than 0.5 L, greater than 1 L, greater than 2 L,greater than 10 L, greater than 250 L, greater than 1000 L, greater than10,000 L, greater than 50,000 L, greater than 100,000 L or greater than200,000 L, and such as when the volume of the aqueous medium is greaterthan 250 L and contained within a steel vessel.

Variously, the carbon source for such culture systems is selected fromdextrose, sucrose, a pentose, a polyol, a hexose, both a hexose and apentose, and combinations thereof, the pH of the aqueous medium is lessthan 7.5, the culture system is aerated, such as at an oxygen transferrate selected from i) greater than 5 mmole/L-hr of oxygen and less than200 mmole/L-hr oxygen; ii) greater than 5 mmole/L-hr of oxygen and lessthan 100 mmole/L-hr oxygen; iii) greater than 5 mmole/L-hr of oxygen andless than 80 mmole/L-hr oxygen; and iv) greater than 5 mmole/L-hr ofoxygen and less than 50 mmole/L-hr oxygen.

In various embodiments, the invention is an aqueous broth obtained froma culture system, wherein said aqueous broth comprises i) aconcentration of 3-hydroxypropionate selected from greater than 5 g/L,greater than 10 g/L, greater than 15 g/L, greater than 20 g/L, greaterthan 25 g/L, greater than 30 g/L, greater than 35 g/L, greater than 40g/L, greater than 50 g/L, greater than 60 g/L, greater than 70 g/L,greater than 80 g/L, greater than 90 g/L, or greater than 100 g/L3-hydroxypropionate; and ii) a concentration of 1,3-propanediol selectedfrom less than 30 g/L; less than 20 g/L; less than 10 g/L; less than 5g/L; less than 1 g/L; or less than 0.5 g/L. In some aspects, the aqueousbroth comprises an amount of biomass selected from less than 20 gDCW/Lbiomass, less than 15 gDCW/L biomass, less than 10 gDCW/L biomass, lessthan 5 gDCW/L biomass or less than 1 gDCW/L biomass. Alternatively, theaqueous broth according to the invention is such that the 3-HP/succinateratio (g3-HP/g succinate) is greater than 3, greater than 10 greaterthan 30, greater than 60, greater than 100, greater than 150 or greaterthan 200. In various aspects, the 3-HP/fumarate ratio (g3-HP/g fumarase)is greater than 3, greater than 10 greater than 30, greater than 60,greater than 100, greater than 150 or greater than 200, or the3-HP/glycerol ratio (g3-HP/g glycerol) is greater than 3, greater than10, greater than 30, Beater than 60, greater than 100, greater than 150or greater than 200, or the 3-HP/acetate ratio (g3-HP/g acetate) isgreater than 1.5, greater than 3, greater than 10, greater than 30,greater than 60, greater than 100, greater than 150 or greater than 200,or the 3-HP/alanine ratio (g3-HP/g alanine) is greater than 3, greaterthan 10, greater than 30, greater than 60, greater than 100, greaterthan 150 or greater than 200, or the 3-HP/beta-alanine ratio (g3-HP/gbeta-alanine) is greater than 1.5, greater than 3, greater than 10,greater than 30, greater than 60, greater than 100, greater than 150 orgreater than 200, or the 3-HP/glutamate ratio (g3-HP/g glutamate) isgreater than 3, greater than 10, greater than 30, greater than 60,greater than 100, greater than 150 or greater than 200, or the3-HP/glutamine ratio (g3-HP/g glutamine) is greater than 3, greater than10, greater than 30, greater than 60, greater than 100, greater than 150or greater than 200, or the 3-HP/3-hydroxypropionaldehyde ratio (g3-HP/g3-hydroxypropioaldehyde) is greater than 1.5, greater than 3, greaterthan 10, greater than 30, greater than 60, greater than 100, greaterthan 150 or greater than 200, or the 3-HP/1,3-propanediol ratio(g′3-HP/g 1,3-propanediol) is greater than 1.5, greater than 3, greaterthan 10, greater than 30, greater than 60, greater than 100, greaterthan 150 or greater than 200, and/or the 3-HP/ lactate ratio (g3-HP/glactate) is greater than 3, greater than 10, greater than 30, greaterthan 60, greater than 100, greater than 150 or greater than 200.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims. A better understanding of the features and advantages of thepresent invention will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanyingdrawings of which:

FIG. 1 depicts metabolic pathways of a microorganism related to aspectsof the present invention, more particularly related to 3-HP production,with gene names of E. coli shown at certain enzymatic steps, the latterfor example and not meant to be limiting.

FIG. 2A depicts metabolic pathways of a microorganism related to aspectsof the present invention, with gene names of E. coli shown at certainenzymatic steps, the latter for example and not meant to be limiting.

FIG. 2B provides a more detailed depiction of representative enzymaticconversions and exemplary E. coli genes of the fatty acid synthetasesystem that was more generally depicted in FIG. 2A.

FIG. 3 provides an exemplary multiple sequence alignment, comparingcarbonic anhydrase polypeptides (CLUSTAL 2.0.12 multiple sequencealignment of Carbonic Anhydrase Polypeptides).

FIG. 4A provides an exemplary sequence alignment: Comparison of DNAsequences of fabIts (JP1111 (SEQ ID No.:769)) and wildtype (BW25113 (SEQID No.:827)) E. coli fabI genes DNA mutation: C722T.

FIG. 4B provides an exemplary sequence alignment: Comparison of proteinsequences of fabI^(Ts) ((JP1111 (SEQ ID No.:770) and wildtype (BW25113(SEQ ID No.:828)) E. coli fabI genes Amino Acid-S241F.

FIGS. 5, 6 and 7 provide data and results from Example 11.

FIG. 8 depicts metabolic pathways of a microorganism with multiplegenetic modifications related to aspects of the present invention, moreparticularly related to 3-HP production, with gene names of E. colishown at certain enzymatic steps, the latter for example and not meantto be limiting.

FIGS. 9A-9G pertain to E. coli 3-HP toleragenic complex (3HPTGC). A)General schematic of FIGS. 9B-G. B) Group F, portion of metabolicpathways, showing pathway products and enzymes that together comprisethe 3HPTGC in E. coli. C) Group A, portion metabolic pathways, showingpathway products and enzymes that together comprise the 3HPTGC in E.coli. D) Group B, portion of metabolic pathways, showing pathwayproducts and enzymes that together comprise the 3HPTGC in E. coli. E)Group C, portion of metabolic pathways, showing pathway products andenzymes that together comprise the 3HPTGC in E. coli. F) Group D,portion of metabolic pathways, showing pathway products and enzymes thattogether comprise the 3HPTGC in E. coli. G) Group E, portion ofmetabolic pathways, showing pathway products and enzymes that togethercomprise the 3HPTGC in E. coli.

FIGS. 9H-9N pertain to Bacillus subtilis 3HPTGC. H) General schematic ofFIGS. 9I-N. Group F, portion of metabolic pathways, showing pathwayproducts and enzymes that together comprise 3HPTGC in B. subtilis. J)Group A, portion metabolic pathways, showing pathway products andenzymes that together comprise the 3HPTGC in B. subtilis. K) Group B,portion of metabolic pathways, showing pathway products and enzymes thattogether comprise the 3HPTGC in B. subtilis. L) Group C, portion ofmetabolic pathways, showing pathway products and enzymes that togethercomprise the 3HPTGC in B. subtilis. M) Group D, portion of metabolicpathways, showing pathway products and enzymes that together comprisethe 3HPTGC in B. subtilis. N) Group E, portion of metabolic pathways,showing pathway products and enzymes that together comprise the 3HPTGCin B. subtilis.

FIGS. 9O-9U pertain to Saccharomyces cerevisiae 3HPTGC. O) Generalschematic of FIGS. 9P-U. P) Group F, portion of metabolic pathways,showing pathway products and enzymes that together comprise the 3HPTGCin S. cerevisiae. Q) Group A, portion metabolic pathways, showingpathway products and enzymes that together comprise the 3HPTGC in S.cerevisiae. R) Group B, portion of metabolic pathways, showing pathwayproducts and enzymes that together comprise the 3HPTGC in S. cerevisiae.S) Group C, portion of metabolic pathways, showing pathway products andenzymes that together comprise the 3HPTGC in S. cerevisiae. T) Group D,portion of metabolic pathways, showing pathway products and enzymes thattogether comprise the 3HPTGC in S. cerevisiae. U) Group E, portion ofmetabolic pathways, showing pathway products and enzymes that togethercomprise the 3HPTGC in S. cerevisiae.

FIGS. 9V-9BB pertain to Cupriavidus necator 3HPTGC. V) General schematicof FIGS. 9W-BB. W) Group F, portion of metabolic pathways, showingpathway products and enzymes that together comprise the 3HPTGC in C.necator. X) Group A, portion metabolic pathways, showing pathwayproducts and enzymes, that together comprise the 3HPTGC in C necator Y)Group B, portion of metabolic pathways, showing pathway products andenzymes that together comprise the 3HPTGC in C. necator. Z) Group C,portion of metabolic pathways, showing pathway products and enzymes thattogether comprise the 3HPTGC in C. necator. AA) Group D, portion ofmetabolic pathways, showing pathway products and enzymes that togethercomprise the 3HPTGC in C. necator. BB) Group E, portion of trietabolicpathways showing pathway products and enzymes that together comprise the3HPTGC in C. necator.

FIG. 10 provides a representation of the glycine cleavage pathway.

FIG. 11 provides, from a prior art reference, a summary of a known 3-HPproduction pathway from glucose to pyruvate to acetyl-CoA to malonyl-CoAto 3-HP.

FIG. 12 provides, from a prior art reference, a summary of a known 3-HPproduction pathway from glucose to phosphoenolpynivate (PEP) tooxaloacetate (directly or via pyruvate) to aspartate to β-alanine tomalonate semialdehyde to 3-HP.

FIG. 13 provides, from a prior art reference, a summary of known 3-HPproduction pathways.

FIGS. 14A and B provide a schematic diagram of natural mixedfermentation pathways in E. coli.

FIG. 15A-P provides graphic data of control microorganisms responses to3-HP; 15A-O are graphic data of control microorganisms and FIG. 15Pprovides a comparison with one genetic modification of the 3HPTGC.

FIG. 16 depicts a known chemical reaction catalyzed byalpha-ketoglutarate encoded by the kgd gene from M. tuberculosis.

FIG. 17 depicts a new enzymatic function, the decarboxylation ofoxaloacetate to malonate semialdehyde that is to be achieved bymodification of the kgd gene.

FIG. 18 shows a proposed selection approach for kgd mutants.

FIG. 19 shows a screening protocol related to the proposed selectionapproach depicted in FIG. 18, FIG. 19A, FIG. 19B and FIG. 19C arevariants of the screening protocol.

FIG. 20 provides a comparison regarding the IroK peptide sequence.

FIG. 21 provides a calibration curve for 3-HP conducted with HPLC.

FIG. 22 provides a calibration curve for 3-HP conducted for GC/MS.

FIG. 23 provides a representative standard curve for the enzymatic assayfor 3-HP.

FIGS. 24A, B, and C and FIGS. 25A and B show a schematic of the entireprocess of converting biomass to a finished product such as a diaper.

Tables also are provided herein and are part of the specification.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to various production methods and/orgenetically modified microorganisms that have utility for fermentativeproduction of various chemical products, to methods of making suchchemical products that utilize populations of these microorganisms invessels, and to systems for chemical production that employ thesemicroorganisms and methods. Among the benefits of the present inventionis increased specific productivity when such microorganisms produce achemical product during a fermentation event or cycle. The presentinvention provides production techniques and/or genetically modifiedmicroorganisms to produce a chemical product of interest, such as3-hydroxypropionic acid (3-HP) with one or more means for modulatingconversion of malonyl-CoA to fatty acyl molecules (which thereafter maybe converted to fatty acids, for example fatty acyl-ACP molecules),wherein the production pathway comprises an enzymatic conversion stepthat uses malonyl-CoA as a substrate. The means for modulatingconversion of malonyl-CoA to fatty acyl molecules, such as fatty acylACP molecules, is effective to balance carbon flow to microbial biomasswith carbon flow to chemical product, and surprisingly affordsachievement of elevated specific productivity rates.

As noted herein, various aspects of the present invention are directedto a microorganism cell comprises a metabolic pathway from malonyl-CoAto 3-HP, and means for modulating conversion of malonyl-CoA to fattyacyl molecules (which thereafter may be converted to fatty acids) alsoare provided. Then, when the means for modulating modulate to decreasesuch conversion, a proportionally greater number of malonyl-CoAmolecules are 1) produced and/or 2) converted via the metabolic pathwayfrom malonyl-CoA to 3-HP. In various embodiments, additional geneticmodifications may be made, such as to 1) increase intracellularbicarbonate levels, such as by increasing carbonic anhydrase, 2)increase enzymatic activity of acetyl-CoA carboxylase, andNADPH-dependent transhydrogenase.

Unexpected increases in specific productivity by a population of agenetically modified microorganism nay be achieved in methods andsystems in which that microorganism has a microbial production pathwayfrom malonyl-CoA to a selected chemical product as well as a reductionin the enzymatic activity of a selected enzyme of the microorganism'sfatty acid synthase system (more particularly, its fatty acid elongationenzymes). In various embodiments, specific supplements to a bioreactorvessel comprising such microorganism population may also he provided tofurther improve the methods and systems.

Additionally, for one chemical product, 3-hydroxypropionic acid (3-HP),genetic modifications for production pathways are provided, and atoleragenic complex is described for which genetic modifications, and/orculture system modifications, limy be made to increase microorganismtolerance to 3-HP. Moreover, genetic modifications to increaseexpression and/or enzymatic activity of carbonic anhydrase and/orcyanase nay provide dual-functions to advantageously improve both 3-HPproduction and 3-HP tolerance.

Other additional genetic modifications are disclosed herein for variousembodiments.

Definitions

As used in the specification and the claims, the singular forms “a,”“an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to an “expressionvector” includes a single expression vector as well as a plurality ofexpression vectors, either the same (e.g., the same operon) ordifferent; reference to “microorganism” includes a single microorganismas well as a plurality of microorganisms; and the like.

As used herein, dry cell weight (DCW) for E. coli strains is calculatedas 0.33 times the measured OD₆₀₀ value, based on baseline DCW to OD₆₀₀determinations.

As used herein, “reduced enzymatic activity,” “reducing enzymaticactivity,” and the like is meant to indicate that a microorganismcell's, or an isolated enzyme, exhibits a lower level of activity thanthat measured in a comparable cell of the same species or its nativeenzyme. That is, enzymatic conversion of the indicated substrate(s) toindicated product(s) under known standard conditions for that enzyme isat least 10, at least 20, at least 30, at least 40, at least 50. atleast 00, at least 70, at least 80, or at least 90 percent less than theenzymatic activity for the same biochemical conversion by a native(non-modified) enzyme under a standard specified condition. This termalso can include elimination of that enzymatic activity. A cell havingreduced enzymatic activity of an enzyme can be identified using anymethod known in the art. For example, enzyme activity assays can be usedto identify cells having reduced enzyme activity. See, for example,Enzyme Nomenclature, Academic Press, Inc., New York 2007.

The term “heterologous DNA,” “heterologous nucleic acid sequence,” thelike as used herein refers to a nucleic acid sequence wherein at leastone of the following is true: (a) the sequence of nucleic acids isforeign to (i.e., not naturally found in) a given host microorganism;(b) the sequence may be naturally found in a given host microorganism,but in an unnatural (e.g., greater than expected) amount; or (c) thesequence of nucleic acids comprises two or more subsequences that arenot found in the same relationship to each other in nature. For example,regarding instance (c), a heterologous nucleic acid sequence that isrecombinantly produced will have two or more sequences from unrelatedgenes arranged to make a new functional nucleic acid.

The term “heterologous” is intended to include the term “exogenous” asthe latter term is generally used in the art. With reference to the hostmicroorganism's genome prior to the introduction of a heterologousnucleic acid sequence, the nucleic acid sequence that codes for theenzyme is heterologous (whether or not the heterologous nucleic acidsequence is introduced into that genome).

As used herein, the term “gene disruption,” or grammatical equivalentsthereof and including “to disrupt enzymatic function,” “disruption ofenzymatic function,” and the like), is intended to mean a geneticmodification to a microorganism that renders the encoded gene product ashaving a reduced polypeptide activity compared with polypeptide activityin or from a microorganism cell not so modified. The geneticmodification can be, for example, deletion of the entire gene, deletionor other modification of a regulatory sequence required fortranscription or translation, deletion of a portion of the gene whichresults in a truncated gene product (e.g., enzyme) or by any of variousmutation strategies that reduces activity (including to no detectableactivity level) the encoded gene product. A disruption may broadlyinclude a deletion of all or part of the nucleic acid sequence encodingthe enzyme, and also includes, but is not limited to other types ofgenetic modifications, e.g., introduction of stop codons, frame shiftmutations, introduction or removal of portions of the gene, andintroduction of a degradation signal, those genetic modificationsaffecting mRNA transcription levels and/or stability, and altering thepromoter or repressor upstream of the gene encoding the enzyme.

In various contexts, a gene disruption is taken to mean any geneticmodification to the DNA, mRNA encoded from the DNA, and thecorresponding amino acid sequence that results in reduced polypeptideactivity. Many different methods can be used to make a cell havingreduced polypeptide activity. For example, a cell can be engineered tohave a disrupted regulatory sequence or polypeptide-encoding sequenceusing common mutagenesis or knock-out technology. See, e.g., Methods inYeast Genetics (1997 edition), Adams et al., Cold Spring Harbor Press(1998). One particularly useful method of gene disruption is completegene deletion because it reduces or eliminates the occurrence of geneticreversions in the genetically modified microorganisms of the invention.Accordingly, a disruption of a gene whose product is an enzyme therebydisrupts enzymatic function. Alternatively, antisense technology can beused to reduce the activity of a particular polypeptide. For example, acell can be engineered to contain a cDNA that encodes an antisensemolecule that prevents a polypeptide from being translated. Further,gene silencing can be used to reduce the activity of a particularpolypeptide.

The term “antisense molecule” as used herein encompasses any nucleicacid molecule or nucleic acid analog (e.g., peptide nucleic acids) thatcontains a sequence that corresponds to the coding strand of anendogenous polypeptide. An antisense molecule also can have flankingsequences (e.g., regulatory sequences). Thus, antisense molecules can beribozymes or anti sense oligonucleotides.

As used herein, a ribozyme can have any general structure including,without limitation, hairpin hammerhead, or axhead structures, providedthe molecule cleaves RNA.

The term “reduction” or “to reduce” when used in such phrase and itsgrammatical equivalents are intended to encompass a complete eliminationof such conversion(s).

Bio-production, as used herein, may be aerobic, microaerobic, oranaerobic.

As used herein, the language “sufficiently homologous” refers toproteins or portions thereof that have amino acid sequences that includea minimum number of identical or equivalent amino acid residues whencompared to an amino acid sequence of the amino acid sequences providedin this application (including the SEQ ID Nos./sequence listings) suchthat the protein or portion thereof is able to achieve the respectiveenzymatic reaction and/or other function. To determine whether aparticular protein or portion thereof is sufficiently homologous may bedetermined by an assay of enzymatic activity, such as those commonlyknown in the art.

Descriptions and methods for sequence identity and homology are intendedto be exemplary and it is recognized that these concepts arewell-understood in the art. Further, it is appreciated that nucleic acidsequences may be varied and still encode an enzyme or other polypeptideexhibiting a desired functionality, and such variations are within thescope of the present invention.

Further to nucleic acid sequences, “hybridization” refers to the processin which two single-stranded polynucleotides bind non-covalently to forma stable double-stranded polynucleotide. The term “hybridization” mayalso refer to triple-stranded hybridization. The resulting (usually)double-stranded polynucleotide is a “hybrid” or “duplex.” “Hybridizationconditions” will typically include salt concentrations of less thanabout 1M, more usually less than about 500 mM and less than about 200mM. Hybridization temperatures can be as low as 5° C., but are typicallygreater than 22° C., more typically greater than about 30° C., and oftenare in excess of about 37° C. Hybridizations are usually performed understringent conditions, i.e. conditions under which a probe will hybridizeto its target subsequence. Stringent conditions are sequence-dependentand are different in different circumstances. Longer fragments mayrequire higher hybridization temperatures for specific hybridization. Asother factors may affect the stringency of hybridization, including basecomposition and length of the complementary strands, presence of organicsolvents and extent of base mismatching, the combination of parametersis more important than the absolute measure of any one alone. Generally,stringent conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH. Exemplarystringent conditions include salt concentration of at least 0.01 M to nomore than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3and a temperature of at least 25° C. For example, conditions of 5×SSPE(750 nM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of25-30° C. are suitable for allele-specific probe hybridizations. Forstringent conditions, see for example, Sambrook and Russell and Anderson“Nucleic Acid Hybridization” 1^(st) Ed., BIOS Scientific PublishersLimited (1999), which are hereby incorporated by reference forhybridization protocols. “Hybridizing specifically to” or “specificallyhybridizing to” or like expressions refer to the binding, duplexing, orhybridizing of a molecule substantially to or only to a particularnucleotide sequence or sequences under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA.

The term “identified enzymatic functional variant” means a polypeptidethat is determined to possess an enzymatic activity and specificity ofan enzyme of interest but which has an amino acid sequence differentfrom such enzyme of interest. A corresponding “variant nucleic acidsequence” may be constructed that is determined to encode such anidentified enzymatic functional variant. For a particular purpose, suchas increased tolerance to 3-HP via genetic modification to increaseenzymatic conversion at one or more of the enzymatic conversion steps ofthe 3HPTGC in a microorganism, one or more genetic modifications may bemade to provide one or more heterologous nucleic acid sequence(s) thatencode one or more identified 3HPTGC enzymatic functional variant(s).That is, each such nucleic acid sequence encodes a polypeptide that isnot exactly the known polypeptide of an enzyme of the 3HPTGC, but whichnonetheless is shown to exhibit enzymatic activity of such enzyme. Suchnucleic acid sequence, and the polypeptide it encodes, may not fallwithin a specified limit of homology or identity yet by its provision ina cell nonetheless provide for a desired enzymatic activity andspecificity. The ability to obtain such variant nucleic acid sequencesand identified enzymatic functional variants is supported by recentadvances in the states of the art in bioinformatics and proteinengineering and design, including advances in computational, predictiveand high-throughout methodologies. Functional variants more generallyinclude enzymatic functional variants, and the nucleic acids sequencesthat encode them, as well as variants of non-enzymatic polypeptides,wherein the variant exhibits the function of the original (target)sequence.

The use of the phrase “segment of interest” is meant to include both agene and any other nucleic acid sequence segment of interest. Oneexample of a method used to obtain a segment of interest is to acquire aculture of a microorganism, where that microorganism's genome includesthe gene or nucleic acid sequence segment of interest.

When the genetic modification of a gene product, i.e., an enzyme, isreferred to herein, including, the claims, it is understood that thegenetic modification is of a nucleic acid sequence, such as or includingthe gene, that normally encodes the stated gene product, i.e., theenzyme.

In some embodiments a truncated respective polypeptide has at leastabout 90% of the full length of a polypeptide encoded by a nucleic acidsequence encoding the respective native enzyme, and more particularly atleast 95% of the full length of a polypeptide encoded by a nucleic acidsequence encoding the respective native enzyme. By a polypeptide havingan amino acid sequence at least, for example, 95% “identical” to areference amino acid sequence of a polypeptide is intended that theamino acid sequence of the claimed polypeptide is identical to thereference sequence except that the claimed polypeptide sequence caninclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of the polypeptide. In other words, to obtain apolypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence can be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence can be inserted into the referencesequence. These alterations of the reference sequence can occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. In other embodimentstruncation may be more substantial, as described elsewhere herein.

Species and other phylogenic identifications are according to theclassification known to a person skilled in the art of microbiology,

Where methods and steps described herein indicate certain eventsoccurring in certain order, those of ordinary skill in the art willrecognize that the ordering of certain steps may be modified and thatsuch modifications are in accordance with the variations of theinvention. Additionally, certain steps may be performed concurrently ina parallel process when possible, as well as performed sequentially.

Prophetic examples provided herein are meant to be broadly exemplary andnot limiting in any way. This applies to the examples regardingseparation and purification of 3-HP, and conversions of 3-HP todownstream compounds, since there are numerous possible approaches tosuch steps and conversions, including those disclosed in referencesrecited and incorporated herein.

The meaning of abbreviations is as follows: “C” means Celsius or degreesCelsius, as is clear from its usage, DCW means dry cell weight, “s”means second(s), “min” means minute(s), “h,” “hr,” or “hrs” meanshour(s), “psi” means pounds per square inch, “nm” means nanometers, “d”means day(s), “μL” or “uL”er “ul” means microliter(s), “mL” meansmilliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” meansnanometers, “mM” means millimolar, “μM” or “uM” means micromolar, “M”means molar, “mmol” means millimole(s), “μmol” or “uMol” meansmicromole(s)”, “g” means gram(s), “μg” or “ug” means microgram(s) and“ng” means nanogram(s), “PCR” means polymerase chain reaction, “OD”means optical density, “OD₆₀₀” means the optical density measured at aphoton wavelength of 600 nm, “kDa” means kilodaltons, “g” means thegravitation constant, “bp” means base pair(s), “kbp” means kilobasepair(s), “% w/v” means weight/volume percent, “% v/v” meansvolume/volume percent, “IPTG” meansisopropyl-μ-D-thiogalactopyranoiside, “RBS” means ribosome binding site,“rpm” means revolutions per minute, “HPLC” means high performance liquidchromatography, and “GC” means gas chromatography. As disclosed herein,“3-HP” means 3-hydroxypropionic acid and “3HPTGC” means the 3-HPtoleragenic complex. Also, 10̂5 and the like are taken to mean 10⁵ andthe like.

I. Carbon Sources

Bio-production media, which is used in the present invention withrecombinant microorganisms having a biosynthetic pathway for 3-HP, mustcontain suitable carbon sources or substrates for the intended metabolicpathways. Suitable substrates may include, but are not limited to,monosaccharides such as glucose and fructose, oligosaccharides such aslactose or sucrose, polysaccharides such as starch or cellulose ormixtures thereof and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, carbon monoxide, or methanol forwhich metabolic conversion into key biochemical intermediates has beendemonstrated. In addition to one and two carbon substratesmethylotrophic organisms are also known to utilize a number of othercarbon containing compounds such as methylamine, glucosamine and avariety of amino acids for metabolic activity.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable in the present invention asa carbon source, common carbon substrates used as carbon sources areglucose, fructose, and sucrose, as well as mixtures of any of thesesugars. Other suitable substrates include xylose, arabinose, othercellulose-based C-5 sugars, high-fructose corn syrup, and various othersugars and sugar mixtures as are available commercially. Sucrose may beobtained from feedstocks such as sugar cane, sugar beets, cassava,bananas or other fruit, and sweet sorghum. Glucose and dextrose may beobtained through saccharification of starch based feedstocks includinggrains such as corn, wheat, rye, barley, and oats. Also, in someembodiments all or a portion of the carbon source may be glycerol.Alternatively, glycerol may be excluded as an added carbon source.

In one embodiment, the carbon source is selected from glucose, fructose,sucrose, dextrose, lactose, glycerol, and mixtures thereof. Variously,the amount of these components in the carbon source may be greater thanabout 50%, greater than about 60%, greater than about 70%, greater thanabout 80%, greater than about 90%, or more, up to 100% or essentially100% of the carbon source.

In addition, methylotrophic organisms are known to utilize a number ofother carbon containing compounds such as methylamine, glucosamine and avariety of amino acids far metabolic activity. For example,methylotrophic yeast are known to utilize the carbon from methylamine toform trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd.(Int. Symp.), 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly,Don P. Publisher: intercept, Andover, UK). Similarly, various species ofCandida will metabolize alanine or oleic acid (Sulter et al., Arch.Microbial. 153:485-489 (1990)). Hence it is contemplated that the sourceof carbon utilized in embodiments of the present invention may encompassa wide variety of carbon-containing substrates.

In addition, fermentable sugars may be obtained from cellulosic andlignocellulosic biomass through processes of pretreatment andsaccharification, as described, for example, in U.S. Patent PublicationNo. 2007/0031918A1, which is herein incorporated by reference. Biomassrefers to any cellulosic or lignocellulosic material and includesmaterials comprising cellulose, and optionally further comprisinghemicellulose, lignin, starch, oligosaccharides and/or monosaccharides.Biomass may also comprise additional components, such as protein and/orlipid. Biomass may be derived from a single source, or biomass cancomprise a mixture derived from more than one source; for example,biomass could comprise a mixture of corn cobs and corn stover, or amixture of grass and leaves. Biomass includes, but is not limited to,bioenergy crops, agricultural residues, municipal solid waste,industrial solid waste, sludge from paper manufacture, yard waste, woodand forestry waste. Examples of biomass include, but are not limited to,corn grain, corn cobs, crop residues such as corn husks, corn stover,grasses, wheat, wheat straw, barley, barley straw, hay, rice straw,switchgrass, waste paper, sugar cane bagasse, sorghum, soy, componentsobtained from milling of grains, trees, branches, roots, leaves, woodchips, sawdust, shrubs and bushes, vegetables, fruits, flowers andanimal manure. Any such biomass may be used in a bio-production methodor system to provide a carbon source. Various approaches to breakingdown cellulosic biomass to mixtures of more available and utilizablecarbon molecules, including sugars, include: heating in the presence ofconcentrated or dilute acid (e.g., <1% sulfuric acid); treating withammonia; treatment with ionic salts; enzymatic degradation; andcombinations of these. These methods normally follow mechanicalseparation and milling, and are followed by appropriate separationprocesses.

In various embodiments, any of a wide range of sugars, including, butnot limited to sucrose, glucose, xylose, cellulose or hemicellulose, areprovided to a microorganism, such as in an industrial system comprisinga reactor vessel in which a defined media (such as a minimal salts mediaincluding but not limited to M9 minimal media, potassium sulfate minimalmedia, yeast synthetic minimal media and many others or variations ofthese), an inoculum of a microorganism providing one or more of the 3-HPbiosynthetic pathway alternatives, and the a carbon source may becombined. The carbon source enters the cell and is cataboliized bywell-known and common metabolic pathways to yield common metabolicintermediates, including phosphoenolpyruvate (PEP). (See MolecularBiology of the Cell, 3rd Ed., B. Alberts et al. Garland Publishing, NewYork, 1994, pp. 42-45, 66-74, incorporated by reference for theteachings of basic metabolic catabolic pathways for sugars; Principlesof Biochemistry, 3rd Ed., D. L. Nelson & M. M. Cox, Worth Publishers,New York, 2000, pp 527-658, incorporated by reference for the teachingsof major metabolic pathways; and Biochemistry, 4th Ed., L. Stryer, W. H.Freeman and Co., New York, 1995, pp. 463-650, also incorporated byreference for the teachings of major metabolic pathways.)

Bio-based carbon can be distinguished from petroleum-based carbonaccording, to a variety of methods, including without limitation ASTMD6866, or various other techniques. For example, carbon-14 and carbon-12ratios differ in bio-based carbon sources versus petroleum-basedsources, where higher carbon-14 ratios are found in bio-based carbonsources. In various embodiments, the carbon source is notpetroleum-based, or is not predominantly petroleum based. In variousembodiments, the carbon source is greater than about 50% non-petroleumbased, greater than about 60% non-petroleum based, greater than about70% non-petroleum based, greater than about 80% non-petroleum based,greater than about 90% non-petroleum based, or more. In variousembodiments, the carbon source has a carbon-14 to carbon-12 ratio ofabout 1.0×10⁻¹⁴ or greater.

Various components may be excluded from the carbon source. For example,in some embodiments, acrylic acid, 1,4-butanediol, and/or glycerol areexcluded or essentially excluded from the carbon source. As such, thecarbon source according to some embodiments of the invention may be lessthan about 50% glycerol, less than about 40% glycerol, less than about30% glycerol, less than about 20% glycerol, less than about 10%glycerol, less than about 5% glycerol, less than about 1% glycerol, orless. For example, the carbon source may be essentially glycerol-free.By essentially glycerol-free is meant that any glycerol that may bepresent in a residual amount does not contribute substantially to theproduction of the target chemical compound.

II. Microorganisms

Features as described and claimed herein may be provided in amicroorganism selected from the listing herein, or another suitablemicroorganism, that also comprises one or more natural, introduced, orenhanced 3-HP bio-production pathways. Thus, in some embodiments themicroorganism comprises an endogenous 3-HP production pathway (whichmay, in some such embodiments, be enhanced), whereas in otherembodiments the microorganism does not comprise an endogenous 3-HPproduction pathway.

Varieties of these genetically modified microorganisms may comprisegenetic modifications and/or other system alterations as may bedescribed in other patent applications of one or more of the presentinventor(s) and/or subject to assignment to the owner of the presentpatent application.

The examples describe specific modifications and evaluations to certainbacterial and yeast microorganisms. The scope of the invention is notmeant to be limited to such species, but to be generally applicable to awide range of suitable Microorganisms. Generally, a microorganism usedfor the present invention may be selected from bacteria, cyanobacteria,filamentous fungi and yeasts.

For some embodiments, microbial hosts initially selected for 3-HPtoleragenic bio-production should also utilize sugars including glucoseat a high rate. Most microbes are capable of utilizing, carbohydrates.However, certain environmental microbes cannot utilize carbohydrates tohigh efficiency, and therefore would not be suitable hosts for suchembodiments that are intended for glucose or other carbohydrates as theprincipal added carbon source.

As the genomes of various species become known, the present inventioneasily may be applied to an ever-increasing range of suitablemicroorganisms. Rather, given the relatively low cost of geneticsequencing, the genetic sequence of a species of interest may readily hedetermined to make application of aspects of the present invention morereadily obtainable (based on the ease of application of geneticmodifications to an organism having a known genomic sequence).

More particularly, based on the various criteria described herein,suitable microbial hosts for the bio-production of 3-HP that comprisetolerance aspects provided herein generally may include, but are notlimited to, any gram negative organisms, more particularly a member ofthe family Enterobacteriaceae, such as E. coli, or Oligotrophacarboxidovorans, or Pseudomononas sp.; any gram positive microorganism,for example Bacillus subtilis, Lactohaccilus sp. or Lactococcus sp.; ayeast, for example Saccharomyces cerevisiae, Pichia pastoris or Pichiastipitis; and other groups or microbial species. More particularly,suitable microbial hosts for the bio-production of 3-HP generallyinclude, but are not limited to, members of the genera Clostridium,Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus,Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus,Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida,Hansenula and Saccharomyces. Hosts that may be particularly of interestinclude: Oligotropha carboxidovorans (such as strain OM5), Escherichiacoli, Alcaligenes eutrophus (Cupriavidus necator), Bacilluslicheniformis, Paenibacillus macerans, Rhodococcus etythropolis,Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium,Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis andSaccharomyces cerevisiae.

More particularly, suitable microbic hosts for the bio-production 3-HPgenerally include, but are not limited to, members of the generaClostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus,Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes,Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansentila and Saccharomyces.

Hosts that may be particularly of interest include: Oligotrophacarboxidovorans (such as strain OM5^(T)), Escherichia coli, Alcaligeneseutrophus (Cupriavidus necator), Bacillus licheniformis, Paenibacillusmacerans, Rhodococcus erythropolis, Pseudomonas Lactobacillus plantarum,Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis,Bacillus subtilis and Saccharomyces cerevisiae. Also, any of the knownstrains of these species may be utilized as a starting microorganism, asmay any of the following species including respective strainsthereof—Cupriavidus basilensis, Cupriavidus campinensis, Cupriavidusgilardi, Cupriavidus lahursis, Cupriavidus metallidurans, Cupriavidusoxalaticus, Cupriavidus pauculus, Cupriavidus pinatubonensis,Cupriavidus respiraculi, and Cupriavidus anensis.

In some embodiments, the recombinant microorganism is a gam-negativebacterium. In some embodiments, the recombinant microorganism isselected from the genera Zymomonas, Escherichia Pseudomonas,Alcaligenes, and Klebsiella. In some embodiments, the recombinantmicroorganism is selected from the species Escherichia coli, Cupriavidusnecator, Oligotropha carboxidovorans, and Pseudomonas putida. In someembodiments, the recombinant microorganism is an E. coli strain.

In some embodiments, the recombinant microorganism is a gram-positivebacterium. In some embodiments, the recombinant microorganism isselected from the genera Clostridium, Salmonella, Rhodococcus, Bacillus,Lactobacillus, Enterococcus, Paenibacillus, Arthrobacter,Corynebacterium, and Brevibacterium. In some embodiments, therecombinant microorganism is selected from the species Bacilluslicheniformis, Paenibacillus macerans, Rhodococcus erythropolis,Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium,Enterococcus faecalis, and Bacillus subtilis. In particular embodiments,the recombinant microorganism is a B. subtilis strain.

In some embodiments, the recombinant microorganism is a yeast, In someembodiments, the recombinant microorganism is selected from the generaPichia, Candida, Hansenula and Saccharomyces. In particular embodiments,the recombinant microorganism is Saccharomyces cerevisiae.

It is further appreciated, in view of the disclosure, that any of theabove microorganisms may be used for production of chemical productsother than 3-HP.

The ability to genetically modify the host is essential for theproduction of any recombinant microorganism. The mode of gene transfertechnology may be by electroporation, conjugation, transduction ornatural transformation. A broad range of host conjugative plasmids anddrug resistance markers are available. The cloning vectors are tailoredto the host organisms based on the nature of antibiotic resistancemarkers that can function in that host.

III. Media and Culture Conditions

In addition to an appropriate carbon source, such as selected from oneof the herein-disclosed types, bio production media must containsuitable minerals, salts, cofactors, buffers and other components, knownto those skilled in the art, suitable for the growth of the cultures andpromotion of the enzymatic pathway necessary for 3-HP production, orother products made under the present invention.

Another aspect of the invention regards media and culture conditionsthat comprise genetically modified microorganisms of the invention andoptionally supplements.

Typically cells are grown at a temperature in the range of about 25° C.to about 40° C. in an appropriate medium, as well as up to 70° C. forthermophilic microorganisms. Suitable growth media in the presentinvention are common commercially prepared media such as Luria Bertani(LB) broth, M9 minimal media, Sabouraud Dextrose (SD) broth, Yeastmedium (YM) broth, (Ymin) yeast synthetic minimal media, and minimalmedia as described herein, such as M9 minimal media. Other defined orsynthetic growth media may also be used, and the appropriate medium forgrowth of the particular microorganism will be known by one skilled inthe art of microbiology or bio-production science. In variousembodiments a minimal media may be developed and used that does notcomprise, or that has a low level of addition of various components, forexample less than 10, 5, 2 or 1 g/L of a complex nitrogen sourceincluding but not limited to yeast extract, peptone, tryptone, soyflour, corn steep liquor, or casein. These minimal medias may also havelimited supplementation of vitamin mixtures including biotin, vitaminB12 and derivatives of vitamin B12, thiamin, pantothenate and othervitamins. Minimal medias may also have limited simple inorganic nutrientsources containing less than 28, 17, or 2.5 mM phosphate, less than 25or 4 mM sulfate, and less than 130 or 50 mM total nitrogen.

Bio-production media, which is used in embodiments of the present withgenetically modified microorganisms, must contain suitable carbonsubstrates for the intended metabolic pathways. As describedhereinbefore, suitable carbon substrates include carbon monoxide, carbondioxide, and various monomeric and oligomeric sugars.

Suitable pH ranges for the bio-production are between pH 3.0 to pH 10.0,where pH 6.0 to pH 8.0 is a typical pH range for the initial condition.However, the actual culture conditions for a particular embodiment arenot meant to be limited by these pH ranges.

Bio-productions may be performed under aerobic, microaerobic, oranaerobic conditions, with or without agitation.

The amount of 3-HP or other product(s) produced in a bio-productionmedia generally can be determined using a number of methods known in theart, for example, high performance liquid chromatography (HPLC), gaschromatography (GC), or GC/Mass Spectroscopy (MS). Specific HPLC methodsfor the specific examples are provided herein.

IV. Bio-Production Reactors and Systems

Fermentation systems utilizing methods and/or compositions according tothe invention are also within the scope of the invention.

Any of the recombinant microorganisms as described and/or referred toherein may be introduced into an industrial bio-production system wherethe microorganisms convert a carbon source into 3-HP in a commerciallyviable operation. The bio-production system includes the introduction ofsuch a recombinant microorganism into a bioreactor vessel, with a carbonsource substrate and bin production media suitable for growing therecombinant microorganism, and maintaining the bio-production systemwithin a suitable temperature range (and dissolved oxygen concentrationrange if the reaction is aerobic or microaerobic) for a suitable time toobtain a desired conversion of a portion of the substrate molecules to3-HP. Industrial bio-production systems and their operation arewell-known to those skilled in the arts of chemical engineering andbioprocess engineering.

Bio-productions may be performed under aerobic, microaerobic, oranaerobic conditions, with or without agitation. The operation ofcultures and populations of microorganisms to achieve aerobic,microaerobic and anaerobic conditions are known in the art, anddissolved oxygen levels of a liquid culture comprising a nutrient mediaand such microorganism populations may be monitored to maintain orconfirm a desired aerobic, microaerobic or anaerobic condition. Whensyngas is used as a feedstock, aerobic, microaerobic, or anaerobicconditions may be utilized. When sugars are used, anaerobic, aerobic ormicroaerobic conditions can be implemented in various embodiments.

Any of the recombinant microorganisms as described and/or referred toherein may be introduced into an industrial bio-production system wherethe microorganisms convert a carbon source into 3-HP, and optionally invarious embodiments also to one or more downstream compounds of 3-HP ina commercially viable operation. The bio-production system includes theintroduction of such a recombinant microorganism into a bioreactorvessel, with a carbon source substrate and bio-production media suitablefor growing the recombinant microorganism, and maintaining thebio-production system within a suitable temperature range (and dissolvedoxygen concentration range if the reaction is aerobic or microaerobic)for a suitable time to obtain a desired conversion of a portion of thesubstrate molecules to 3-HP.

In various embodiments, syngas components or sugars are provided to amicroorganism, such as in an industrial system comprising a reactorvessel in which a defined media (such as a minimal salts media includingbut not limited to M9 minimal media, potassium sulfate minimal media,yeast synthetic minimal media and many others or variations of these),an inoculum of a microorganism providing, an embodiment of thebiosynthetic pathway(s) taught herein, and the carbon source may becombined. The carbon source enters the cell and is catabolized bywell-known and common metabolic pathways to yield common metabolicintermediates, including phosphoenolpyruvate (PEP). (See MolecularBiology of the Cell, 3^(rd) Ed., B. Alberts et al. Garland Publishing,New York, 1994, pp. 42-45, 66-74, incorporated by reference for theteachings of basic metabolic catabolic pathways for sugars; Principlesof Biochemistry, 3^(rd) D. L. Nelson & M. M. Cox, Worth Publishers, NewYork, 2000, pp. 527-658, incorporated by reference for the teachings ofmajor metabolic pathways; and Biochemistry, 4^(th) Ed., L. Stryer, W. H.Freeman and Co., New York, 1995, pp. 463-650, also incorporated byreference for the teachings of major metabolic pathways.).

Further to types of industrial bio-production, various embodiments ofthe present invention may employ a hatch type of industrial bioreactor.A classical batch bioreactor system is considered “closed” meaning thatthe composition of the medium is established at the beginning of arespective bio-production event and not subject to artificialalterations and additions during the time period ending substantiallywith the end of the bio-production event. Thus, at the beginning of thebio-production event the medium is inoculated with the desired organismor organism s, and bio-production is permitted to occur without addinganything to the system. Typically, however, a “batch” type ofbio-production event is hatch with respect to the addition of carbonsource and attempts are often made at controlling factors such as pH andoxygen concentration. In hatch systems the metabolite and biomasscompositions of the system change constantly up to the time thebio-production event is stopped. Within batch cultures cells moderatethrough a static lag phase to a high growth log phase and finally to astationary phase where growth rate is diminished or halted. Ifuntreated, cells in the stationary phase will eventually die. Cells inlog phase generally are responsible for the bulk of production of adesired end product or intermediate.

A variation on the standard batch system is the fed-batch system,Fed-batch bio-production processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe nutrients, including the substrate, are added in increments as thebio-production progresses. Fed Batch systems are useful when cataboliterepression is apt to inhibit the metabolism of the cells and where it isdesirable to have limited amounts of substrate in the media. Measurementof the actual nutrient concentration in Fed-Batch systems may bemeasured directly, such as by sample analysis at different times, orestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases such as CO₂.Batch and fed-batch approaches are common and well known in the art andexamples may be found in Thomas D. Brock in Biotechnology: A Textbook ofindustrial Microbiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass., Deshpande, Mukund V., Appl. Biochem. Biotechnol.,36:227, (1992), and Biochemical Engineering Fundamentals, 2^(nd) Ed. J.E. Bailey and D. F. Ollis, McGraw Hill, New York, 1986, hereinincorporated by reference for general instruction on bio-production.

Although embodiments of the present invention may be performed in batchmode, or in fed-batch mode, it is contemplated that the invention wouldbe adaptable to continuous bio-production methods. Continuousbio-production is considered an “open” system where a definedbio-production medium is added continuously to a bioreactor and an equalamount of conditioned media is removed simultaneously for processing.Continuous bio-production generally maintains the cultures within acontrolled density range where cells are primarily in log phase growth.Two types of continuous bioreactor operation include a chemostat,wherein fresh media is fed to the vessel while simultaneously removingan equal rate of the vessel contents. The limitation of this approach isthat cells are lost and high cell density generally is not achievable.In fact, typically one can obtain much higher cell density with afed-hatch process. Another continuous bioreactor utilizes perfusionculture, which is similar to the chemostat approach except that thestream that is removed from the vessel is subjected to a separationtechnique which recycles viable cells back to the vessel. This type ofcontinuous bioreactor operation has been shown to yield significantlyhigher cell densities than fed-batch and can be operated continuously.Continuous bio production is particularly advantageous for industrialoperations because it has less down time associated with draining,cleaning and preparing the equipment for the next bio-production event.Furthermore, it is typically more economical to continuously operatedownstream unit operations, such as distillation, than to run them inbatch mode.

Continuous bio-production allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Methods of modulatingnutrients and growth factors for continuous bio-production processes aswell as techniques for maximizing the rate of product formation are wellknown in the art of industrial microbiology and a variety of methods aredetailed by Brock, supra.

It is contemplated that embodiments of the present invention may bepracticed using either batch, fed-batch or continuous processes and tintany known mode of bio-production would be suitable. It is contemplatedthat cells may be immobilized on an inert scaffold as whole cellcatalysts and subjected to suitable bio-production conditions for 3-HPproduction, or be cultured in liquid media in a vessel, such as aculture vessel. Thus, embodiments used in such processes, and inbio-production systems using these processes, include a population ofgenetically modified microorganisms of the present invention, a culturesystem comprising such population in a media comprising nutrients forthe population, and methods of making 3-HP and thereafter, a downstreamproduct of 3-HP.

Embodiments of the invention include methods of making 3-HP in abio-production system, some of which methods may include obtaining 3-HPafter such bio-production event. For example, a method of making 3-HPmay comprise: providing to a culture vessel a media comprising suitablenutrients; providing to the culture vessel an inoculum of a geneticallymodified microorganism comprising genetic modifications described hereinsuch that the microorganism produces 3-HP from syngas and/or a sugarmolecule; and maintaining the culture vessel under suitable conditionsfor the genetically modified microorganism to produce 3-HP.

It is within the scope of the present invention to produce, and toutilize in bio-production methods and systems, including industrialbio-production systems for production of 3-HP, a recombinantmicroorganism genetically engineered to modify one or more aspectseffective to increase tolerance to 3-HP (and, in some embodiments, also3-HP bio-production) by at least 20 percent over control microorganismlacking the one or more modifications.

In various embodiments, the invention is directed to a system forbioproduction of acrylic acid as described herein, said systemcomprising; a tank for saccharification of biomass; a line for passingthe product of saccharification to a fermentation tank optionally via apre fermentation tank; a fermentation tank suitable for microorganismcell culture; a line for discharging contents from the fermentation tankto an extraction and/or separation vessel; an extraction and/orseparation vessel suitable for removal of 3-hydroxyprobionic acid fromcell culture waste; a line for transferring 3-hydroxypropionic acid to adehydration vessel; and a dehydration vessel suitable for conversion of3-hydroxypropionic acid to acrylic acid. In various embodiments, thesystem includes one or more pre-fermentation tanks, distillationcolumns, centrifuge vessels, back extraction columns, mixing vessels, orcombinations thereof.

The following published resources are incorporated by reference hereinfor their respective teachings to indicate the level of skill in theserelevant arts, and as needed to support a disclosure that teaches how tomake and use methods of industrial bio-production of 3-HP, or otherproduct(s) produced under the invention, from sugar sources, and alsoindustrial systems that may be used to achieve such conversion with anyof the recombinant microorganisms of the present invention (BiochemicalEngineering Fundamentals, 2^(nd) Ed, J. E. Bailey and D. F. Ollis,McGraw Hill, New York, 1986, entire book for purposes indicated andChapter 9, pages 533-657 in particular for biological reactor design;Unit Operations of Chemical Engineering, 5^(th) Ed., W. L. McCabe etal., McGraw Hill, New York 1993, entire hook for purposes indicated, andparticularly for process and separation technologies analyses;Equilibrium Staged Separations, P. C. Wankat, Prentice Hall, EnglewoodCliffs, N.J. USA, 1988, entire book for separation technologiesteachings). Generally, it is further appreciated, in view of thedisclosure, that any of the above methods and systems may be used forproduction of chemical products other than 3-HP.

V. Genetic Modifications, Nucleotide Sequences, and Amino Acid Sequences

Embodiments of the present invention may result from introduction of anexpression vector into a host microorganism, wherein the expressionvector contains a nucleic acid sequence coding far an enzyme that is, oris not, normally found in a host microorganism.

The ability to genetically modify a host cell is essential for theproduction of any genetically modified (recombinant) microorganism. Themode of gene transfer technology may be by electroporation, conjugation,transduction, or natural transformation. A broad range of hostconjugative plasmids and drug resistance markers are available. Thecloning vectors are tailored to the host organisms based on the natureof antibiotic resistance markers that can function in that host. Also,as disclosed herein, a genetically modified (recombinant) microorganismmay comprise modifications other than via plasmid introduction,including modifications to its genomic DNA.

It has long been recognized in the art that some amino acids in aminoacid sequences can be varied without significant effect on the structureor function of proteins. Variants included can constitute deletions,insertions, inversions, repeats, and type substitutions so long as theindicated enzyme activity is not significantly adversely affected.Guidance concerning which amino acid changes are likely to bephenotypically silent can be found, inter aria, in Bowie, J. U., et al.,“Deciphering the Message in Protein Sequences; Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990). This reference isincorporated by reference for such teachings, which are, however, alsogenerally known to those skilled in the art.

In various embodiments polypeptides obtained by the expression of thepolynucleotide molecules of the present invention may have at leastapproximately 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identity to one or more amino acid sequences encoded by the genes and/ornucleic acid sequences described herein for the 3-HP tolerance-relatedand biosynthesis pathways.

As a practical inatter, whether any particular polypeptide is at least50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identicalto any reference amino acid sequence of any polypeptide described herein(which may correspond with a particular nucleic acid sequence describedherein), such particular polypeptide sequence can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set such that the percentage of identity iscalculated over the hill length of the reference amino acid sequence andthat gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

For example, in a specific embodiment the identity between a referencesequence (query sequence, i.e., a sequence of the present invention) anda subject sequence, also referred to as a global sequence alignment, maybe determined using the FASTDB computer program based on the algorithmof Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferredparameters for a particular embodiment in which identity is narrowlyconstrued, used in a FASTDB amino acid alignment, are: ScoringScheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection is made to the results to take into consideration the factthat the FASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating gjobal percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that arelateral to the N- and C-terminal of the subject sequence, which are notmatched/aligned with a corresponding subject residue, as a percent ofthe total bases of the query sequence. A determination of whether aresidue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence are considered for thismanual correction. For example, a 90 amino acid residue subject sequenceis aligned with a 100 residue query sequence to determine percentidentity. The deletion occurs at the N-terminus of the subject sequenceand therefore, the FASTDB alignment does not show a matching/alignmentof the first 10 residues at the N-terminus. The 10 unpaired residuesrepresent 10% of the sequence (number of residues at the N- andC-termini not matched/total number of residues in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched thefinal percent identity would be 90%. In another example, a 90 residuesubject sequence is compared with a 100 residue query sequence. Thistime the deletions are internal deletions so there are no residues atthe N- or C-termini of the subject sequence which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for.

More generally, nucleic acid constructs can be prepared comprising anisolated polynucleotide encoding a polypeptide having enzyme activityoperably linked to one or more (several) control sequences that directthe expression of the coding sequence in a microorganism, such as E.coli, under conditions compatible with the control sequences. Theisolated polynucleotide may be manipulated to provide for expression ofthe polypeptide. Manipulation of the polynucleotide's sequence prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesequences utilizing recombinant DNA methods are well established in theart.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell. Examples of suitablepromoters for directing transcription of the nucleic acid constructs,especially in an E. coli host cell, are the lac promoter (Gronenborn,1976, Mol. Gen. Genet. 148: 243-250), tac promoter (DeBoer et al., 1983,Proceedings of the National Academy of Sciences USA 80: 21-25), trcpromoter (Brosius et al, 1985, J. Biol. Chem. 260: 3539-3541), T7 RNApolymerase promoter (Studier and Moffatt, 1986, J. Mol. Biol. 189:113-130), phage promoter p_(L) (Elvin et al., 1990, Gene 87: 123-126),tetA prmoter (Skena, 1994, Gene 151: 131-135), araBAD promoter (Guzmanet: al., 1995, J. Bacteriol. 177: 4121-4130), and rhaP_(BAD) promoter(Haldimann et al., 1998, J. Bacteriol, 180: 1277-1286). Other promotersare described in “Useful proteins from recombinant bacteria” inScientific American, 1980, 242: 74-94; and in Sambrook and Russell,“Molecular Cloning: A Laboratory Manual,” Third Edition 2001 (volumes1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in an E. coli cell may be used in thepresent invention. It may also be desirable to add regulatory sequencesthat allow regulation of the expression of the polypeptide relative tothe growth of the host cell. Examples of regulatory systems are thosethat cause the expression of the gene to be turned on or off in responseto a chemical or physical stimulus, including the presence of aregulatory compound. Regulatory systems in prokaryotic systems includethe lac, tac, and trp operator systems.

For various embodiments of the invention the genetic manipulations maybe described to include various genetic manipulations, including thosedirected to change regulation of, and therefore ultimate activity of, anenzyme or enzymatic activity of an enzyme identified in any of therespective pathways. Such genetic modifications may be directed totranscriptional, translational, and post-translational modificationsthat result in a change of enzyme activity and/or selectivity underselected and/or identified culture conditions and/or to provision ofadditional nucleic acid sequences such as to increase copy number and/ormutants of an enzyme related to 3-HP production. Specific methodologiesand approaches to achieve such genetic modification are well known toone skilled in the art, and include, but are not limited to: increasingexpression of an endogenous genetic element; decreasing functionality ofa repressor gene; introducing a heterologous genetic element; increasingcopy number of a nucleic acid sequence encoding a polypeptide catalyzingan enzymatic conversion step to produce 3-HP; mutating a genetic elementto provide a mutated protein to increase specific enzymatic activity;over-expressing; under-expressing; over-expressing a chaperone; knockingout a protease; altering or modifying feedback inhibition; providing anenzyme variant comprising one or more of an impaired binding site for arepressor and/or competitive inhibitor; knocking out a repressor gene;evolution, selection and/or other approaches to improve mRNA stabilityas well as use of plasmids having an effective copy number and promotersto achieve an effective level of improvement. Random mutagenesis may bepracticed to provide genetic modifications that may fall into any ofthese or other stated approaches. The genetic modifications furtherbroadly fall into additions (including insertions), deletions (such asby a mutation) and substitutions of one or more nucleic acids in anucleic acid of interest. In various embodiments a genetic modificationresults in improved enzymatic specific activity and/or turnover numberof an enzyme. Without being limited, changes may be measured by one ormore of the following: K_(M); K_(cat); and K_(avidity).

In various embodiments, to function more efficiently, a microorganismmay comprise one or more gene deletions. For example, in E. coli, thegenes encoding the lactate dehydrogenase (IdhA), phosphateacetyltransferase (pta), pyruvate oxidase (poxB), and pyruvate-formatelyase (pflB) may be disrupted, including deleted. Such gene disruptions,including deletions, are not meant to be limiting, and may beimplemented in various combinations in various embodiments. Genedeletions may be accomplished by mutational gene deletion approaches,and/or starting with a mutant strain having reduced or no expression ofone or more of these enzymes, and/or other methods known to thoseskilled in the art. Gene deletions may be effectuated by any of a numberof known specific methodologies, including but not limited to the RED/ETmethods using kits and other reagents sold by Gene Bridges (Gene BridgesGmbH, Dresden, Germany, <<www.genebridges.com>>).

More particularly as to the latter method, use of Red/ET recombination,is known to those of ordinary skill in the art and described in U.S.Pat. Nos. 6,355,412 and 6,509,156, issued to Stewart et al. andincorporated by reference herein for its teachings of this method.Material and kits for such method are available from Gene Bridges (GeneBridges GmbH, Dresden, Germany, <<www.genebridges.com>>), and the methodmay proceed by following the manufacturer's instructions. The methodinvolves replacement of the target gene by a selectable marker viahomologous recombination performed by the recombinase from λ-phage. Thehost organism expressing λ-red recombinase is transformed with a linearDNA product coding for a selectable marker flanked by the terminalregions (generally ˜50 bp, and alternatively up to about ˜300 bp)homologous with the target gene. The marker could then be removed byanother recombination step performed by a plasmid vector carrying theFLP-recombinase, or another recombinase, such as Cre.

Targeted deletion of parts of microbial chromosomal DNA or the additionof foreign genetic material to microbial chromones may be practiced toalter a host cell's metabolism so as to reduce or eliminate productionof undesired metabolic products. This may be used in combination withother genetic modifications such as described herein in this generalexample. In this detailed description, reference has been made tomultiple embodiments and to the accompanying drawings in which is shownby way of illustration specific exemplary embodiments in which theinvention may be practiced. These embodiments are described insufficient detail to enable those skilled in the art to practice theinvention, and it is to be understood that modifications to the variousdisclosed embodiments may be made by a skilled artisan.

Further, for 3-HP production, such genetic modifications may be chosenand/or selected for to achieve a higher flux rate through certainenzymatic conversion steps within the respective 3-HP production pathwayand so may affect general cellular metabolism in fundamental and/ormajor ways.

It will be appreciated that amino acid “homology” includes conservativesubstitutions, i.e. those that substitute a given amino acid in apolypeptide by another amino acid of similar characteristics. Typicallyseen as conservative substitutions are the following replacements:replacements of an aliphatic amino acid such as Ala, Val, Leu and Ilewith another aliphatic amino acid; replacement of a Ser with a Thr orvice versa; replacement of an acidic residue such as Asp or Glu withanother acidic residue; replacement of a residue bearing an amide group,such as Asn or Gln, with another residue bearing an amide group;exchange of a basic residue such as Lys or Arg with another basicresidue; and replacement of an aromatic residue such as Phe or Tyr withanother aromatic residue.

For all nucleic acid and amino acid sequences provided herein, it isappreciated that conservatively modified variants of these sequences areincluded, and are within the scope of the invention in its variousembodiments. Functionally equivalent nucleic acid and amino acidsequences (functional variants), which may include conservativelymodified variants as well as more extensively varied sequences, whichare well within the skill of the person of ordinary skill in the art,and microorganisms comprising these, also are within the scope ofvarious embodiments of the invention, as are methods and systemscomprising such sequences and/or microorganisms. In various embodiments,nucleic acid sequences encoding sufficiently homologous proteins orportions thereof are within the scope of the invention. More generally,nucleic acids sequences that encode a particular amino acid sequenceemployed in the invention may vary due to the degeneracy of the geneticcode, and nonetheless fall within the scope of the invention. Thefollowing table provides a summary of similarities among amino acids,upon which conservative and less conservative substitutions may bebased, and also various codon redundancies that reflect this degeneracy.

TABLE 1 Amino Acid Relationships DNA codons Alanine N, AliGCT, GCC, GCA, GCG Proline N CCT, CCC, CCA, CCG Valine N, AliGTT, GTC, GTA, GTG Leucine N, Ali CTT, CTC, CTA, CTG, TTA, TTGIsoleucine N, Ali ATT, ATC, ATA Methionine N ATG Phenylalanine N, AroTTT, TTC Tryptophan N TCC Glycine PU GGT, GGC, GGA, GGG Serine PUTCT, TCC, TCA, TCG, AGT, AGC Threonine PU ACT, ACC, ACA, ACG AsparaginePU, Ami AAT, AAC Glutamine PU, Ami CAA, CAG Cysteine PU TGT, TGCAspartic NEG, A GAT, GAC acid Glutamic NEG, A GAA, GAG acid ArgininePOS, B CGT, CGC, CGA, CGG, AGA, AGG Lysine POS, B AAA, AAG Histidine POSCAT, CAC Tyrosine Aro TAT, TAC Stop Codons TAA, TAG, TGA

Legend: side groups and other related properties: A=acidic; B=basic;Ali=aliphatic; Ami=amine; Aro=aromatic; N=nonpolar; PU=polar, uncharged;NEG=negatively charged; POS=positively charged.

Also, variants and portions of particular nucleic acid sequences, andrespective encoded amino acid sequences recited herein may be exhibit adesired functionality, e.g., enzymatic activity at a selected level,when such nucleic acid sequence variant and/or portion contains a 15nucleotide sequence identical to any 15 nucleotide sequence set forth inthe nucleic acid sequences recited herein including, without limitation,the sequence starting at nucleotide number 1 and ending at nucleotidenumber 15, the sequence starting at nucleotide number 2 and ending atnucleotide number 16, the sequence starting at nucleotide number 3 andending at nucleotide number 17, and so forth. It will he appreciatedthat the invention also provides isolated nucleic acid that contains anucleotide sequence that is greater than 15 nucleotides (e.g., 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides)in length and identical to any portion of the sequence set forth innucleic acid sequences recited herein. For example, the inventionprovides isolated nucleic acid that contains a 25 nucleotide sequenceidentical to any 25 nucleotide sequence set forth in any one or more(including any grouping of) nucleic acid sequences recited hereinincluding, without limitation, the sequence starting at nucleotidenumber 1 and ending at nucleotide number 25, the sequence starting atnucleotide number 2 and ending at nucleotide number 26, the sequencestarting at nucleotide number 3 and ending at nucleotide number 27, andso forth. Additional examples include, without limitation, isolatednucleic acids that contain a nucleotide sequence that is 50 or morenucleotides (e.g., 100, 150, 200, 250, 300, or more nucleotides) inlength and identical to any portion of any of the sequences disclosedherein. Such isolated nucleic acids can include, without limitation,those isolated nucleic acids containing a nucleic acid sequencerepresented in any one section of discussion and/or examples, such asregarding 3-HP production pathways, nucleic acid sequences encodingenzymes of the fatty acid synthase system, or 3-HP tolerance. Forexample, the invention provides an isolated nucleic acid containing anucleic acid sequence listed herein that contains a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). Such isolatednucleic acid molecules can share at least 60, 65, 70, 75, 80, 85, 90,95, 97, 98, or 99 percent sequence identity with a nucleic acid sequencelisted herein (i.e., in the sequence listing).

Additional examples include, without limitation, isolated nucleic acidsthat contain a nucleic acid sequence that encodes an amino acid sequencethat is 50 or more amino acid residues (e.g., 100, 150, 200, 250, 300,or more amino acid residues) in length and identical to any portion ofan amino acid sequence listed or otherwise disclosed herein.

In addition, the invention provides isolated nucleic acid that containsa nucleic acid sequence that encodes an amino acid sequence having avariation of an amino acid sequence listed or otherwise disclosedherein. For example, the invention provides isolated nucleic acidcontaining a nucleic acid sequence encoding an amino acid sequencelisted or otherwise disclosed herein that contains a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). Such isolatednucleic acid molecules can contain a nucleic acid sequence encoding anamino acid sequence that shares at least 60, 65, 70, 75, 80, 85, 90, 95,97, 98, or 99 percent sequence identity with an amino acid sequencelisted or otherwise disclosed herein.

Examples of properties that provide the bases for conservative and otheramino acid substitutions are exemplified in Table 1. Accordingly, oneskilled in the art may make numerous substitutions to obtain an aminoacid sequence variant that exhibits a desired functionality. BLASTP,CLUSTALP, and other alignment and comparison tools may be used to assesshighly conserved regions, to which fewer substitutions may be made(unless directed to alter activity to a selected level, which mayrequire multiple substitutions). More substitutions may be made inregions recognized or believed to not be involved with an active site orother binding or structural motif. In accordance with Table 1, forexample, substitutions may be made of one polar uncharged (P1.1) aminoacid for a polar uncharged amino acid of a listed sequence, optionallyconsidering size/molecular weight (i.e., substituting a serine for athreonine). Guidance concerning which amino acid changes are likely tobe phenotypically silent can be found, inter alia, in Bowie, J. U., et:Al., “Deciphering the Message in Protein Sequences: Tolerance to AminoAcid Substitutions,” Science 247:1306-1310 (1990). This reference isincorporated by reference for such teachings, which are, however, alsogenerally known to those skilled in the art. Recognized conservativeamino acid substitutions comprise (substitutable amino acids followingeach colon of a set): ala:ser; arg:lys; asn:gln or his; asp:glu;cys:ser; gln:asn; glu:asp; gly:pro; his:asn or gln; ile:leu or val;leu:ile or val; lys:arg or gln or glu; met:leu or ile; phe:met or leu ortyr; ser:thr; thr:ser; trp:tyr; tyr:trp or phe; val:ile or leu.

It is noted that codon preferences and codon usage tables for aparticular species can be used to engineer isolated nucleic acidmolecules that take advantage of the codon usage preferences of thatparticular species. For example, the isolated nucleic acid providedherein can be designed to have codons that are preferentially used by aparticular organism of interest. Numerous software and sequencingservices are available for such codon-optimizing of sequences.

The invention provides polypeptides that contain the emitre amino acidsequence of an amino acid sequence listed or otherwise disclosed herein.In addition, the invention provides polypeptides that contain a portionof an amino acid sequence listed or otherwise disclosed herein. Forexample, the invention provides polypeptides that contain a 15 aminoacid sequence identical to any 15 amino acid sequence of an amino acidsequence listed or otherwise disclosed herein including, withoutlimitation, the sequence starting at amino acid residue number 1 andending at amino acid residue number 15, the sequence starting at aminoacid residue number 2 and ending at amino acid residue number 16, thesequence starting at amino acid residue number 3 and ending at aminoacid residue number 17, and so forth. It will be appreciated that theinvention also provides polypeptides that contain an amino acid sequencethat is greater than 15 amino acid residues (e.g., 16, 17, 15, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues) inlength and identical to any portion of an amino acid sequence listed orotherwise disclosed herein For example, the invention providespolypeptides that contain a 25 amino acid sequence identical to any 25amino acid sequence of an amino acid sequence listed or otherwisedisclosed herein including, without limitation, the sequence starting atamino acid residue number 1 and ending at amino acid residue number 25,the sequence starting at amino acid residue number 2 and ending at aminoacid residue number 26, the sequence starting at amino acid residuenumber 3 and ending at amino acid residue number 27, and so forth.Additional examples include, without limitation, polypeptides thatcontain an amino acid sequence that is 50 or more amino acid residues(e.g., 100, 150, 200, 250, 300 or more amino acid residues) in lengthand identical to any portion of an amino acid sequence listed orotherwise disclosed herein. Further, it is appreciated that, per above,a 15 nucleotide sequence will provide a 5 amino acid sequence, so thatthe latter, and higher-length amino acid sequences, may be defined bythe above-described nucleotide sequence lengths having identity with asequence provided herein.

In addition, the invention provides polypeptides that an amino acidsequence having a variation of the amino acid sequence set forth in anammo acid sequence Fisted or otherwise disclosed herein. For example,the invention provides polypeptides containing an amino acid sequencelisted or otherwise disclosed herein that contains a single insertion, asingle deletion, a single substitution, multiple insertions, multipledeletions, multiple substitutions, or any combination thereof (e.g.,single deletion together with multiple insertions). Such polypeptidescan contain an amino acid sequence that shares at least 60, 65, 70, 75,80, 85, 90, 95, 97, 98 or 99 percent sequence identity with an aminoacid sequence listed or otherwise disclosed herein. A particular variantamino acid sequence may comprise any number of variations as well as anycombination of types of variations.

The invention includes, in various embodiments, an amino acid sequencehaving a variation of any of the polynucleotide and polypeptidesequences disclosed herein. As one example, variations are exemplifiedfor the carbonic anhydrase (E. coli cynT) amino acid sequence set forthin SEQ ID NO:544. FIG. 3 provides a CLUSTAL multiple sequence alignmentof the E. coli carbonic anhydrase aligned with carbonic anhydrases ofeleven other species that had relatively high homology, based on low Evalues, in a BLASTP comparison. SEQ ID NO:544 is the fifth sequenceshown. Multiple conservative and less conservative substitutions areshown (i.e., by the “:” and “.” designations, respectively), which canlead to additional modifications by one skilled in the art. Thus,examples of variations of the sequence set forth in SEQ ID NO:544include, without limitation, any variation of the sequences as set forthin FIG. 3. Such variations are provided in FIG. 3 in that a comparisonof the amino acid residue (or lack thereof) at a particular position ofthe sequence set forth in SEQ ID NO:544 with the amino acid residue (orlack thereof) at the same aligned position of any of the other elevenamino acid sequences of FIG. 3 provides a list of specific changes farthe sequence set forth in SEQ ID NO:544. For example, the “E” glutamicacid at position 14 of SEQ ID NO:544 can be substituted with a “D”aspartic acid or “N” asparagine as indicated in FIG. 3. It will beappreciated that the sequence set forth in SEQ ID NO:544 can contain anynumber of variations as well as any combination of types of variations.It is noted that the amino acid sequences provided in FIG. 3 can bepolypeptides having carbonic anhydrase activity.

As indicated herein, polypeptides having a variant amino acid sequencecan retain enzymatic activity. Such polypeptides can be produced bymanipulating the nucleotide sequence encoding a polypeptide usingstandard procedures such as site-directed mutagenesis or various PCRtechniques. As noted herein, one type of modification includes thesubstitution of one or more amino acid residues for amino acid residueshaving a similar chemical and/or biochemical property. For example, apolypeptide can have an amino acid sequence set forth in an amino acidsequence listed or otherwise disclosed herein comprising one or moreconservative substitutions.

More substantial changes can be obtained by selecting substitutions thatare less conservative, and/or in areas of the sequence that may be morecritical, for example selecting residues that differ more significantlyin their effect on maintaining: (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation; (b) the charge or hydrophobicity of thepolypeptide at the target site; or (c) the bulk of the side chain. Thesubstitutions that in general are expected to produce the greatestchanges in polypeptide function are those in which: (a) a hydrophilicresidue, e.g., serine or threonine, is substituted for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine oralanine; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysine, arginine, or histidine, is substituted for (or by) anelectronegative residue, e.g., glutamic acid or aspartic acid; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. The effects ofthese amino acid substitutions (or other deletions or additions) can beassessed for polypeptides having enzymatic activity by analyzing theability of the polypeptide to catalyze the conversion of the samesubstrate as the related native polypeptide to the same product as therelated native polypeptide. Accordingly, polypeptides having 5, 10, 20,30, 40. 50 or less conservative substitutions are provided by theinvention.

Polypeptides and nucleic acids encoding polypeptides can be produced bystandard DNA mutagenesis techniques, for example, M13 primermutagenesis. Details of these techniques are provided in Sambrook andRussell, 2001. Nucleic acid molecules can contain changes of a codingregion to fit the codon usage bias of the particular organism into whichthe molecule is to be introduced.

Alternatively, the coding region can be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence in sucha way that, while the nucleic acid sequence is substantially altered, itnevertheless encodes a polypeptide having an amino acid sequenceidentical or substantially similar to the native amino acid sequence.For example, alanine is encoded in the open reading frame by thenucleotide codon triplet GCT. Because of the degeneracy of the geneticcode, three other nucleotide codon triplets—GCA, GCC, and GCG—also codefor alanine. Thus, the nucleic acid sequence of the open reading framecan be changed at this position to any of these three codons withoutaffecting the amino acid sequence of the encoded polypeptide or thecharacteristics of the polypeptide. Based upon the degeneracy of thegenetic code, nucleic acid variants can be derived from a nucleic acidsequence disclosed herein using standard DNA mutagenesis techniques asdescribed herein, or by synthesis of nucleic acid sequences. Thus, forvarious embodiments the invention encompasses nucleic acid moleculesthat encode the same polypeptide but vary in nucleic acid sequence byvirtue of the degeneracy of the genetic code.

The invention also provides an isolated nucleic acid that is at leastabout 12 bases in length (e.g., at least about 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 40, 50, 60, 100, 250, 500. 750, 1000, 1500, 2000, 3000,4000, or 5000 bases in length) and hybridizes, under hybridizationconditions, to the sense or antisense strand of a nucleic acid having asequence listed or otherwise disclosed herein. The hybridizationconditions can be moderately or highly stringent hybridizationconditions. Also, in some embodiments the microorganism comprises anendogenous 3-HP production pathway (which may, in some such embodiments,be enhanced), whereas in other embodiments the microorganism does notcomprise a 3-HP production pathway, but is provided with one or morenucleic acid sequences encoding polypeptides having enzymatic activityor activities to complete a pathway, described herein, resulting inproduction of 3-HP. In some embodiments, the particular sequencesdisclosed herein, or conservatively modified variants thereof, areprovided to a selected microorganism, such as selected from one or moreof the species and groups of species or other taxonomic groups listedherein.

VI. Redirecting Malonyl-CoA from Fatty Acid Synthesis to 3-HP

Compositions of the present invention, such as genetically modifiedmicroorganisms, comprise a production pathway for a chemical product inwhich malonyl-CoA is a substrate, and may also comprise one or moregenetic modifications to reduce the activity of enzymes encoded by oneor more of the fatty acid synthetase system genes. The compositions maybe used in the methods and systems of the present invention.

Regarding microbial fermentation of a number of chemical products inmany microorganisms of commercial fermentation interest, malonyl-CoA isa metabolic intermediate that, under normal growth conditions, isconverted to fatty acids and derivatives thereof, such as phospholipids,that are then used in cell membranes and for other key cellularfunctions. For example, in Escherichia coil, the fatty acid synthasesystem is a type II or dissociated fatty acid synthase system. In thissystem the enzymes of fatty acid production pathway are encoded bydistinct genes, and, common for many critical metabolic pathways, iswell-regulated, including by downstream products inhibiting upstreamenzymes.

In various microorganisms conversion of the metabolic intermediatemalonyl-CoA to fatty acids via a fatty acid synthesis system (i.e.,pathway or complex) is the only or the major use of malonyl-CoA. It hasbeen determined that when a production pathway to an alternativechemical product exists in a microorganism, reducing such conversion ofmalonyl-CoA to fatty acids can improve metrics for production of thatalternative chemical product (e.g., 3-HP). For example, in manymicroorganism cells the fatty acid synthase system comprisespolypeptides that have the following enzymatic activities:malonyl-CoA-acyl carrier protein (ACP) transacylase; β-ketoacyl-ACPsynthase; β-ketoacyl-ACP reductase; β-hydroxyacyl-ACP dehydratase;3-hydroxyacyl-(acp) dehydratase; and enoyl-acyl carrier proteinreductase (enoyl-ACP reductase). In various embodiments nucleic acidsequences that encode temperature-sensitive forms of these polypeptidesmay be introduced in place of the native enzymes, and when suchgenetically modified microorganisms are cultured at elevatedtemperatures (at which these thermolabile polypeptides becomeinactivated, partially or completely, due to alterations in proteinstructure or complete denaturation), there is observed an increase in aproduct such as 3-HP. In other embodiments other types of geneticmodifications may be made to otherwise modulate, such as lower,enzymatic activities of one or more of these polypeptides. In variousembodiments a result of such genetic modifications is to shiftmalonyl-CoA utilization so that there is a reduced conversion ofmalonyl-CoA to fatty acids, overall biomass, and proportionally greaterconversion of carbon source to a chemical product such as 3-HP. Invarious embodiments, the specific productivity for the microbiallyproduced chemical product is unexpectedly high. Also, additional geneticmodifications, such as to increase malonyl-CoA production, may be madefor certain embodiments.

One enzyme, enoyl(acyl carrier protein) reductase (EC No. 1.3.1.9, alsoreferred to as enoyl-ACP reductase) is a key enzyme for fatty acidbiosynthesis from malonyl-CoA. Escherichia coli this enzyme, FabI, isencoded by the gene fahI (See “Enoyl-Acyl Carrier Protein (fabI) Plays aDeterminant Role in Completing Cycles of Fatty Acid Elongation inEscherichia coli,” Richard J. Heath and Charles O. Rock, J. Biol. Chem.270:44, pp. 26538-26543 (1995), incorporated by reference for itsdiscussion of fabI and the fatty acid synthase system).

The present invention may utilize a microorganism that is provided witha nucleic acid sequence (polynucleotide) that encodes a polypeptidehaving enoyl-ACP reductase enzymatic activity that may be modulatedduring a fermentation event. For example, a nucleic acid sequenceencoding a temperature-sensitive enoyl-ACP reductase may be provided inplace of the native enoyl-ACP reductase, so that an elevated culturetemperature results in reduced enzymatic activity, which then results ina shifting utilization of malonyl-CoA to production of a desiredchemical product. At such elevated temperature the enzyme is considerednon-permissive, as is the temperature. One such sequence is a mutanttemperature-sensitive fabI (fabI^(Ts)) of E. coli, SEQ ID NO:769 forDNA, SEQ ID NO:770 for protein.

It is appreciated that nucleic acid and amino acid sequences forenoyl-ACP reductase in species other than E. coli are readily obtainedby conducting homology searches in known genomics databases, such asBLASTN and BLASTP. Approaches to obtaining homologues in other speciesand functional equivalent sequences are described herein. Accordingly,it is appreciated that the present invention may be practiced by oneskilled in the art for many microorganism species of commercialinterest.

Other approaches than a temperature-sensitive enoyl-ACP reductase may beemployed as known to those skilled in the art, such as, but not limitedto, replacing a native enoyl-ACP or enoyl-coA reductase with a nucleicacid sequence that includes an inducible promoter for this enzyme, sothat an initial induction may be followed by no induction, therebydecreasing enoyl-ACP er enoyl-coA reductase enzymatic activity after aselected cell density is attained.

In some aspects, compositions, methods and systems of the presentinvention shift utilization of malonyl-CoA in a genetic modifiedmicroorganism, which comprises at least one enzyme of the fatty acidsynthase system, such as enoyl-acyl carrier protein reductase (enoyl-ACPreductase) or enoyl-coenzyme A reductase (enoyl-coA reductase),β-ketoacyl-ACP synthase or β-ketoacyl-coA synthase malonyl-CoA-ACP, andmay further comprise at least one genetic modification of nucleic acidsequence encoding carbonic anhydrase to increase bicarbonate levels inthe microorganism cell and/or a supplementation of its culture mediumwith bicarbonate and/or carbonate, and may further comprise one or moregenetic modifications to increase enzymatic activity of one or more ofacetyl-CoA carboxylase and NADPH-dependent transhydrogenase. Moregenerally, addition of carbonate and/or bicarbonate may be used toincrease bicarbonate levels in a fermentation broth.

In some aspects, the present invention comprises a genetically modifiedmicroorganism that comprises at least one genetic modification thatprovides, completes, or enhances a 3-HP production pathway effective toconvert malonyl-CoA to 3-HP, and further comprises a geneticmodification of carbonic anhydrase to increase bicarbonate levels in themicroorganism cell and/or a supplementation of its culture medium withbicarbonate and/or carbonate, and may further comprise one or moregenetic modifications to increase enzymatic activity of one or more ofacetyl-CoA carboxylase and NADPH-dependent transhydrogenase. Relatedmethods and systems utilize such genetically modified microorganism.

In some aspects, the present invention comprises a genetically modifiedmicroorganism that comprises at least one genetic modification thatprovides, completes, or enhances a 3-HP production pathway effective toconvert malonyl-CoA to 3-HP, and further comprises a geneticmodification of at least one enzyme of the fatty acid synthase system,such as enoyl-acyl carrier protein reductase (enoyl-ACP reductase) orenoyl-coenzyme A reductase (enoyl-coA reductase), β-ketoacyl-ACPsynthase or β-ketoacyl-coA synthase, malonyl-CoA-ACP, and may furthercomprise a genetic modification of carbonic anhydrase to increasebicarbonate levels in the microorganism cell and/or a supplementation ofits culture medium with bicarbonate and/or carbonate, and may furthercomprise one or more genetic modifications to increase enzymaticactivity of one or more of acetyl-CoA carboxylase and NADPH-dependenttranshydrogenase. Related methods and systems utilize such geneticallymodified microorganism.

In various embodiments the present invention is directed to a method ofmaking a chemical product comprising: providing a selected cell densityof a genetically modified microorganism population in a vessel, whereinthe genetically modified microorganism comprises a production pathwayfor production of a chemical product from malonyl-CoA; and reducingenzymatic activity of at least one enzyme of the genetically modifiedmicroorganism's fatty acid synthase pathway.

In various embodiments, reducing the enzymatic activity of an enoyl-ACPreductase in a microorganism host cell results in production of 3-HP atelevated specific and volumetric productivity. In still otherembodiments, reducing the enzymatic activity of an enoyl-CoA reductasein a microorganism host cell results in production of 3-HP at elevatedspecific and volumetric productivity.

Another approach to genetic modification to reduce enzymatic activity ofthese enzymes is to provide an inducible promoter that promotes one suchenzyme, such as the enoyl-ACP reductase gene (e.g., fabI in E. coli). Insuch example this promoter may be induced (such as withisopropyl-μ-D-thiogalactopyranoiside (IPTG)) during a first phase of amethod herein, and after the IPTG is exhausted, removed or diluted outthe second step, of reducing enoyl-ACP reductase enzymatic activity, maybegin. Other approaches may be applied to control enzyme expression andactivity such as are described herein and/or known to those skilled inthe art.

While enoyl-CoA reductase is considered an important enzyme of the fattyacid synthase system, genetic modifications may be made to anycombination of the polynucleotides (nucleic acid sequences) encoding thepolypeptides exhibiting the enzymatic activities of this system, such asare listed herein. For example, FabB, β-ketoacyl-acyl carrier proteinsynthase I, is an enzyme in E. coli that is essential for growth and thebiosynthesis of both saturated and unsaturated fatty acids. Inactivationof FabB results in the inhibition of fatty acid elongation anddiminished cell growth as well as eliminating a futile cycle thatrecycles the malonate moiety of malonyl-ACP back to acetyl-CoA. FabF,β-ketoacyl-acyl carrier protein synthase II, is required fir thesynthesis of saturated fatty acids and the control membrane fluidity incells. Both enzymes are inhibited by cerulenin.

It is reported that overexpression of FabF results in diminished fattyacid biosynthesis. It is proposed that FabF outcompetes FabB forassociation with FabD, malonyl-CoA:ACP transacylase. The association ofFabB with FabD is required for the condensation reaction that initiatesfatty acid elongation. (See Microbiological Reviews, September 1993, p.522-542 Vol. 57, No. 3; K. Magnuson et al., “Regulation of Fatty AcidBiosynthesis in Escherichia coli,” American Society for Microbiology; W.Zha et al., “Improving cellular malonyl-CoA level in Escherichia colivia metabolic engineering,” Metabolic Engineering 11 (2009) 192-198). Analternative to genetic modification to reduce such fatty acid synthaseenzymes is to provide into a culture system a suitable inhibitor of oneor more such enzymes. This approach may be practiced independently or incombination with the genetic modification approach. Inhibitors, such ascerulenin, thiotactomycin, and triclosan (this list not limiting) orgenetic modifications directed to reduce activity of enzymes encoded byone or more of the fatty acid synthetase system genes may be employed,singly or in combination.

Without being bound to a particular theory, it is believed that reducingthe enzymatic activity of enoyl-ACP reductase (and/or of other enzymesof the fatty acid synthase system) in a microorganism leads to anaccumulation and/or shunting of malonyl-CoA, a metabolic intermediateupstream of the enzyme, and such malonyl-CoA may then be converted to achemical product for which the microorganism cell comprises a metabolicpathway that utilizes malonyl-CoA. In certain compositions, methods andsystems of the present invention the reduction of enzymatic activity ofenoyl-ACP reductase (or, more generally, of the fatty acid synthasesystem) is made to occur after a sufficient cell density of agenetically modified microorganism is attained. This bi-phasic cultureapproach balances a desired quantity of catalyst, in the cell biomasswhich supports a particular production rate, with yield, which may bepartly attributed to having less carbon be directed to cell mass afterthe enoyl-ACP reductase activity (and/or activity of other enzymes ofthe fatty acid synthase system) is/are reduced. This results in ashifting net utilization of malonyl-CoA, thus providing for greatercarbon flux to a desired chemical product.

In various embodiments of the present invention the specificproductivity is elevated and this results in overall rapid and efficientmicrobial fermentation methods and systems. In various embodiments thevolumetric productivity also is substantially elevated.

In various embodiments a genetically modified microorganism comprises ametabolic pathway that includes conversion of malonyl-CoA to a desiredchemical product, 3-hydroxypropionic acid (3-HP). This is viewed asquite advantageous for commercial 3-HP production economics and isviewed as an advance having clear economic benefit. Other chemicalproducts also are disclosed herein.

The improvements in both specific and volumetric productivity parametersare unexpected and advance the art.

The reduction of enoyl-ACP reductase activity and/or of other enzymes ofthe fatty acid synthase system may be achieved in a number of ways, asis discussed herein.

By “means for modulating” the conversion of malonyl-CoA to fattyacyl-ACP or fatty acyl-coA molecules, and to fatty acid molecules, ismeant any one of the following: 1) providing in a microorganism cell atleast one polynucleotide that encodes at least one polypeptide havingactivity of one of the fatty acid synthase system enzymes (such asrecited herein), wherein the polypeptide so encoded has (such as bymutation and/or promoter substitution, etc., to lower enzymaticactivity), or may be modulated to have (such as by temperaturesensitivity, inducible promoter, etc.) a reduced enzymatic activity; 2)providing to a vessel comprising a microorganism cell or population aninhibitor that inhibits enzymatic activity of one or more of the fattyacid synthase system enzymes (such as recited herein), at a dosageeffective to reduce enzymatic activity of one or more of these enzymes.These means may be provided in combination with one another. When ameans for modulating involves a conversion, during a fermentation event,from a higher to a lower activity of the fatty acid synthetase system,such as by increasing temperature of a culture vessel comprising apopulation of genetically modified microorganism comprising atemperature-sensitive fatty acid synthetase system polypeptide (e.g.,enoyl-ACP reductase), or by adding an inhibitor, there are conceived twomodes—one during which there is higher activity, and a second duringwhich there is lower activity, of such fatty acid synthetase system.During the lower activity mode, a shift to greater utilization ofmalonyl-CoA to a selected chemical product may proceed.

Once the modulation is in effect to decrease the noted enzymaticactivity(ies), each respective enzymatic activity so modulated may bereduced by at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, or at least 90 percentcompared with the activity of the native, non-modulated enzymaticactivity (such as in a cell or isolated). Similarly, the conversion ofmalonyl-CoA to fatty acyl-ACP or fatty acyl-coA molecules may be reducedby at least 10, at least 20, at least 30, at least 40, at least 50, atleast 60, at least 70, at least 80, or at least 90 percent compared withsuch conversion in a non-modulated cell or other system. Likewise, theconversion of malonyl-CoA to fatty acid molecules may be reduced by atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, or at least 90 percent compared with suchconversion in a non-modulated cell or other system.

VII. Production Pathway from Malonyl-CoA to 3-HP

In various embodiments the compositions, methods and systems of thepresent invention involve inclusion of a metabolic production pathwaythat converts malonyl-CoA to a chemical product of interest.

As one example, 3-HP is selected as the chemical product of interest.

Further as to specific sequences for 3-HP production pathway,malonyl-CoA reductase (mcr) from C. aurantiacus was gene synthesized andcodon optimized by the services of DNA 2.0. The FASTA sequence is shownin SEQ ID NO:783 (gi|42561982|gb|AAS20429.1| malonyl-CoA reductase(Chloroflexus aurantiacus)).

Mcr has very few sequence homologs in the NCBI data base. Blast searchesfinds 8 different sequences when searching over the entire protein.Hence development of a pile-up sequences comparison is expected to yieldlimited information. However, embodiments of the present inventionnonetheless may comprise any of these eight sequences, shown herein andidentified as SEQ NOs:784 to 791, which are expected to be but are notyet confirmed to be bi-functional as to this enzymatic activity. Otherembodiments may comprise mutated and other variant forms of any of SEQID NOs:784 to 791, as well as polynucleotides (including, variant formswith conservative and other substitutions), such as those introducedinto a selected microorganism to provide or increase 3-HP productiontherein.

The portion of a CLUSTAL 2.0.11 multiple sequence alignment identifiesthese eight sequences with respective SEQ ID NOs: 783-791, as shown inthe following table.

TABLE 2 Seq ID Reference Nos. No. Genus Speciesgi|42561982|gb|AAS20429.1 783 Chloroflexus aurantiacusgi|163848165|ref|YP_001636209 784 Chloroflexus aurantiacus J-10-flgi|219848167|ref|YP_002462600 785 Chloroflexus aggregans DSM 9485gi|156742880|ref|YP_001433009 786 Roseiflexus castenholzii DSM 13941gi|148657307|ref|YP_001277512 787 Roseiflexus sp. RS-1gi|85708113|ref|ZP_01039179.1 788 Erythrobacter sp. NAP1gi|254282228|ref|ZP_04957196.1 789 gamma proteobacterium NOR51-Bgi|254513883|ref|ZP_05125944.1 790 gamma proteobacterium NOR5-3gi|119504313|ref|ZP_01626393.1 791 3marine gamma proteobacterium HTCC208

Malonyl-CoA may be converted to 3-HP in a microorganism that comprisesone or more of the following:

A bi-functional malonyl-CoA reductase, such as may be obtained fromChloroflexus aurantiacus and other microorganism species. Bybi-functional in this regard is meant that the malonyl-CoA reductasecatalyzes both the conversion of malonyl-CoA to malonate semialdehyde,and of malonate semialdehyde to 3-HP.

A mono-functional malonyl-CoA reductase in combination with a 3-HPdehydrogenase. By mono-functional is meant that the malonyl-CoAreductase catalyzes the conversion of malonyl-CoA to malonatesemialdehyde.

Any of the above polypeptides may be NADH- or NADPH-dependent, andmethods known in the art may be used to convert a particular enzyme tobe either form. More particularly, as noted in WO 2002/042418, “anymethod can be used to convert a polypeptide that uses NADPH as acofactor into a polypeptide that uses NADH as a cofactor such as thosedescribed by others (Eppink et al:, J Mol. Biol., 292 (1): 87-96 (1999),Hall and Tomsett, Microbiology, 146 (Pt 6): 1399-406 (2000), and Dohr etal., Proc. Natl. Acad. Sci., 98 (1): 81-86 (2000).”

Without being limiting, a bi-functional malonyl-CoA reductase limy beselected front the malonyl-CoA reductase of Chloroflexus aurantiacus(such as from ATCC 29365) and other sequences. Also without beinglimiting, a mono-functional malonyl-CoA reductase may be selected fromthe malonyl-CoA reductase of Sulfolobus tokodaii (SEQ ID NO:826). As tothe malonyl-CoA reductase of C. aurantiacus, that sequence and otherspecies' sequences may also be bi-functional as to this enzymaticactivity.

When a mono-functional malonyl-CoA reductase is provided in amicroorganism cell, 3-HP dehydrogenase enzymatic activity also may beprovided to convert malonate semialdehyde to 3-HP. As shown in theexamples, a mono-functional malonyl-CoA reductase may be obtained bytruncation of a bi-functional mono-functional malonyl CoA, and combinedin a strain with an enzyme that converts malonate semialdehyde to 3-HP.

Also, it is noted that another malonyl-CoA reductase is known inMetalloshaera sedula (Msed_709, identified as malonyl-CoAreductase/succinyl-CoA reductase).

By providing nucleic acid sequences that encode polypeptides having theabove enzymatic activities, a genetically modified microorganism maycomprise an effective 3-HP pathway to convert malonyl-CoA to 3-HP inaccordance with the embodiments of the present invention.

Other 3-HP pathways, such as those comprising an aminotransferase (see,e.g., WO 2010/011874, published Jan. 28, 2010), may also he provided inembodiments of a genetically modified microorganism of the presentinvention.

Incorporated into this section, the present invention provides forelevated specific and volumetric productivity metrics as to productionof a selected chemical product, such as 3-hydroxypropionic acid (3-HP).In various embodiments, production of a chemical product, such as 3-HP,is not linked to growth.

In various embodiments, production of 3-HP, or alternatively one of itsdownstream products such as described herein, may reach at least 1, atleast 2, at least 5, at least 10, at least 20, at least 30, at least 40,and at least 50 g/liter titer, such as by using one of the methodsdisclosed herein.

As may be realized by appreciation of the advances disclosed herein asthey relate to commercial fermentations of selected chemical products,embodiments of the present invention may be combined with other geneticmodifications and/or method or system modulations so as to obtain amicroorganism (and corresponding method) effective to produce at least10, at least 20, at least 30, at least 40, at least 45, at least 50, atleast 80, at least 100, or at least 120 grams of a chemical product,such as 3-HP, per liter of final (e.g., spent) fermentation broth whileachieving this with specific and/or volumetric productivity rates asdisclosed herein.

In some embodiments a microbial chemical production event (i.e., afermentation event using a cultured population of a microorganism)proceeds using a genetically modified microorganism as described herein,wherein the specific productivity is between 0.01 and 0.60 grams of 3-HPproduced per gram of microorganism cell on a dry weight basis per hour3-HP/g DCW-hr). In various embodiments the specific productivity isgreater than 0.01, greater than 0.05, greater than 0.10, greater than0.15, greater than 0.20, greater than 0.25, greater than 0.30, greaterthan 0.35, greater than 0.40, greater than 0.45, or greater than 0.50 g3-HP/g DCW-hr. Specific productivity may be assessed over a 2, 4, 6, 8.12 or 24 hour period in a particular microbial chemical productionevent. More particularly, the specific productivity for 3-HP or otherchemical product is between 0.05 and 0.10, 0.10 and 0.15, 0.15 and 0.20,0.20 and 0.25, 0.25 and 0.30, 0.30 and 0.35, 0.35 and 0.40, 0.40 and0.45, or 0.45 and 0.50 g 3-HP/g DCW-hr., 0.50 and 0.55, or 0.55 and 0.60g 3-HP/g DCW-hr. Various embodiments comprise culture systemsdemonstrating such productivity.

Also, in various embodiments of the present invention the volumetricproductivity achieved may be 0.25 g 3-HP (or other chemical product) perliter per hour (g (chemical product)/L-hr), may be greater than 0.25 g3-HP (or other chemical product)/L-hr, may be greater than 0.50 g 3-HP(or other chemical product)/L-hr, may be greater than 1.0 g 3-HP (orother chemical product)/L-hr, may be greater than 1.50 g 3-HP (or otherchemical product)/L-hr, may be greater than 2.0 g 3-HP (or otherchemical product)/L-hr, may be greater than 2.50 g 3-HP (or otherchemical product)/L-hr, may be greater than 3.0 g 3-HP (or otherchemical product)/L-hr, may be greater than 3.50 g 3-HP (or otherChemical product)/L-hr, may be greater than 4.0 g 3-HP (or otherchemical product)/L-hr, may be greater than 4.50 g 3-HP (or otherchemical product)/L-hr, may be greater than 5.0 g 3-HP (or otherchemical product)/L-hr, may be greater than 5.50 g 3-HP (or otherchemical product)/L-hr, nay be greater than 6.0 g 3-HP (or otherchemical product)/L-hr, may be greater than 6.50 g 3-HP (or otherchemical product)/L-hr, may be greater than 7.0 g 3-HP (or otherchemical product)/L-hr, may be greater than 7.50 g 3-HP (or otherchemical product)/L-hr, may be greater than 8.0 g (or other chemicalproduct)/L-hr, may be greater than 8.50 g 3-HP (or other chemicalproduct)/L-hr, may be greater than 9.0 g 3-HP (or other chemicalproduct)/L-hr, may be greater than 9.50 g 3-HP (or other chemicalproduct)/L-hr, or may be greater than 10.0 g 3-HP (or other chemicalproduct)/L-hr.

In some embodiments, specific productivity as measured over a 24-hourfermentation (culture) period may be greater than 0.01, 0.05, 0.10,0,20, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 or12.0 grams of chemical product per gram DCW of microorganisms (based onthe final DCW at the end of the 24-hour period).

In various aspects and embodiments of the present invention, there is aresulting substantial increase in microorganism specific productivitythat advances the fermentation art and commercial economic feasibilityof microbial chemical production, such as of 3-HP (but not limitedthereto).

Stated in another manner, in various embodiments the specificproductivity exceeds (is at least) 0.01 g chemical product/g DCW-hr,exceeds (is at least) 0.05 g chemical product/g DCW-hr, exceeds (is atleast) 0.10 g chemical product/g DCW-hr, exceeds (is at least) 0.15 gchemical product/g DCW-hr, exceeds (is at least) 0.20 g chemicalproduct/g DCW-hr, exceeds (is at least) 0.25 g chemical product/gDCW-hr, exceeds (is at least) 0.30 g chemical product/g DCW-hr, exceeds(is at least) 0.35 g chemical product/g DCW-hr, exceeds (is at least)0.40 g chemical product/g DCW-hr, exceeds (is at least) 0.45 g chemicalproduct/g DCW-hr, exceeds (is at least) 0.50 g chemical product/gDCW-hr, exceeds (is at least) 0.60 g, chemical product/g DCW-hr.

More generally, based on various combinations of the geneticmodifications described herein, optionally in combination withsupplementations described herein, specific productivity values for3-HP, and for other chemical products described herein, may exceed 0.01g chemical product/g DCW-hr, may exceed 0.05 g chemical product/gDCW-hr, may exceed 0.10 g chemical product/g DCW-hr, may exceed 0.15 gchemical product/g DCW-hr, may exceed 0.20 g chemical product/g DCW-hr,may exceed 0.25 g chemical product/g DCW-hr, may exceed 0.30 g chemicalproduct/g DCW-hr, may exceed 0.35 g chemical product/g DCW-hr, mayexceed 0.40 g chemical product/g DCW-hr, may exceed 0.45 g chemicalproduct/g DCW-hr, and may exceed 0.50 g or 0.60 chemical product/gDCW-hr. Such specific productivity may be assessed over a 2, 4, 6, 8, 12or 24 hour period in a particular microbial chemical production event.

The improvements achieved by embodiments of the present invention may bedetermined by percentage increase in specific productivity, or bypercentage increase in volumetric productivity, compared with anappropriate control microorganism lacking the particular geneticmodification combinations taught herein (with or without the supplementstaught herein, added to a vessel comprising the microorganismpopulation). For particular embodiments and groups thereof, suchspecific productivity and/or volumetric productivity improvements is/areat least 10, at least 20, at least 30, at least 40, at least 50, atleast 100, at least 200, at least 300, at least 400, and at least 500percent over the respective specific productivity and/or volumetricproductivity of such appropriate control microorganism.

The specific methods and teachings of the specification, and/or citedreferences that are incorporated by reference, may be incorporated intothe examples. Also, production of 3-HP, or one of its downstreamproducts such as described herein, may reach at least 1, at least 2, atleast 5, at least 10, at least 20, at least 30, at least 40, and atleast 50 g/liter titer in various embodiments.

The metrics may be applicable to any of the compositions, e.g.,genetically modified microorganisms, methods, e.g., of producing 3-HP orother chemical products, and systems, e.g., fermentation systemsutilizing the genetically modified microorganisms and/or methodsdisclosed herein.

It is appreciated that iterative improvements using the strategies andmethods provided herein, and based on the discoveries of theinterrelationships of the pathways and pathway portions, may lead toeven greater 3-HP production and tolerance and more elevated 3-HP titersat the conclusion of a 3-HP bio-production event.

Any number of strategies may lead to development of a suitable modifiedenzyme suitable for use in a 3-HP production pathway. With regard tomalonyl-CoA-reductase, one may utilize or modify an enzyme such asencoded by the sequences in the table immediately above, to achieve asuitable level of 3-HP production capability in a microorganism strain.

VIII. Increasing Tolerance to 3-HP

A complex comprising all or portions of a number of inter-relatedmetabolic pathways has been identified, wherein genetic modification toincrease enzymatic activities of enzymes of such complex, named the 3-HPToleragenic Complex (“3HPTGC”), are demonstrated to increasemicroorganism tolerance to exposure to 3-HP. The 3HPTGC is described inWO 2010/011874, published Jan. 28, 2010, which is incorporated in thepresent application for its teachings of the 3HPTGC and combinations ofgenetic modifications related to 3HP production and tolerance based onthe 3HPTGC and groups therein.

As described and detailed herein, the present invention broadly relatesto alterations, using, genetic modifications, and/or medium modulations(e.g. additions of enzymatic conversion products or other specificchemicals), to achieve desired results in microbe-based industrialbio-production methods, systems and compositions. As to the toleranceaspects, this invention flows from the discovery of the unexpectedimportance of the 3HPTGC which comprises certain metabolic pathwayportions comprising enzymes whose increased activity (based onincreasing copy numbers of nucleic acid sequences that encode there)correlates with increased tolerance of a microorganism to 3-HP.

Actual data and/or prophetic examples directed to alterations of the3HPTGC are provided herein. These examples are intended to demonstratethe breadth of applicability (based on the large number of genomicelements related to the 3HPTGC that demonstrate increased 3-HPtolerance) and some specific approaches to achieve increased toleranceto 3-HP. Approaches may be combined to achieve additive or synergisticimprovements in 3-HP tolerance, and may include alterations that aregenetic or non-genetic (e.g., relating to system supplementation withparticular chemicals, or general alterations to the industrial system).In addition, specific production strategies are disclosed andexemplified.

Thus, in addition to the above-described genetic modifications, directedto providing a 3-HP production pathway and to providing a nucleic acidsequence comprising and/or controlling a gene encoding an enoyl-ACPreductase that allows for control of enzymatic activity of the latterenzyme, and/or as described herein other modifications of the fatty acidsynthetase system, in various embodiments one or more geneticmodifications may be made to the genetically modified microorganism toincrease its tolerance to 3-HP (or other chemical products).

Accordingly, in some embodiments of the present invention, a geneticallymodified microorganism may comprise at least one genetic modification toprovide, complete, or enhance one or more 3-HP production pathways, atleast one genetic modification to provide enoyl-ACP reductase enzymaticactivity and/or other modifications of the fatty acid synthetase systemthat can be controlled so as to reduce such activity at a desired celldensity, and at least one genetic modification of the 3HPTGC, or one,two, or three or more groups thereof, to increase tolerance of thegenetically modified microorganism to 3-HP.

Accordingly, one aspect of the invention relates to a geneticallymodified microorganism comprising at least one genetic modificationeffective to increase 3-hydroxypropionic acid (“3-HP”) production,wherein the increased level of 3-HP production is greater than the levelof 3-HP production in the wild-type microorganism, and at least onegenetic modification of a metabolic complex identified herein as the3-HP Toleragenic Complex (“3HPTGC”). Under certain conditions, such asculture in minimal media, the 3HPTGC genetic modification(s) allow thegenetically modified microorganism to produce 3-HP under specificculture conditions such that 3-HP may accumulate to a relatively higherconcentration without the toxic effects observed in unmodifiedmicroorganisms. The at least one genetic modification of a 3-HPproduction pathway may be to improve 3-HP accumulation and/or productionof a 3-HP production pathway found in the wild-type microorganism, ormay be to provide sufficient enzymatic conversions in a microorganismthat normally does not synthesize 3-HP so that 3-HP is thusbio-produced. Methods of making such genetically modified microorganismsalso are described and are part of this aspect of the invention.

Another aspect of the invention relates to a genetically modifiedmicroorganism comprising at least one genetic modification from two ormore of the chorismate, threonine/homocysteine, polyamine synthesis,lysine synthesis, and nucleotide synthesis portions of the 3HPTGC.Non-limiting examples of multiple combinations exemplify the advantagesof this aspect of the invention. Additional genetic modificationspertain to other portions of the 3HPTGC. Capability to bio-produce 3-HPmay be added to some genetically modified microorganisms by appropriategenetic modification. Methods of identifying genetic modifications toprovide a microorganism achieving an increased 3-HP tolerance, andmicroorganisms made by such methods, relate to this aspect of theinvention.

Another aspect of the invention relates to a genetically modifiedmicroorganism that is able to produce 3-hydroxypropionic acid (“3-HP”),comprising at least one genetic modification to the 3HPTGC thatincreases enzymatic conversion at one or more enzymatic conversion stepsof the 3HPTGC for the microorganism, and wherein the at least onegenetic modification increases 3-HP tolerance of the geneticallymodified microorganism above the 3-HP tolerance of a controlmicroorganism lacking the genetic modification. Methods of making suchgenetically modified microorganisms also are described and are part ofthis aspect of the invention.

Another aspect of the invention relates to a genetically modifiedmicroorganism comprising various core sets of specific geneticmodification(s) of the 3HPTGC. In various embodiments this aspect mayadditionally comprise at least one genetic modification from one or moreor two or more of the chorismate, threonine/homocysteine, polyaminesynthesis, lysine synthesis, and nucleotide synthesis portions of the3HPTGC. Methods of making such genetically modified microorganisms alsoare described and are part of this aspect of the invention.

Further, the invention includes methods of use to improve amicroorganism's tolerance to 3-HP, which may be in a microorganismhaving 3-HP production capability (whether the latter is naturallyoccurring, enhanced and/or introduced by genetic modification).

Also, another aspect of the invention is directed to providing one ormore supplements, which are substrates (i.e., reactants) and/or productsof the 3HPTGC (collectively herein “products” noting that substrates ofall but the initial conversion steps are also products of the 3HPTGC),to a culture of a microorganism to increase the effective tolerance ofthat microorganism to 3-HP.

Another aspect of the invention regards the genetic modification tointroduce a genetic element that encodes a short polypeptide identifiedherein as IroK. The introduction of genetic elements encoding this shortpolypeptide has been demonstrated to improve 3-HP tolerance in E. coliunder microaerobic conditions. This genetic modification may be combinedwith other genetic modifications and/or supplement additions of theinvention.

As to methods of making 3-HP in accordance with the teachings of thisinvention, and to genetically modified microorganisms that make 3-HP,one or more genetic modifications may be provided to a microorganism toincrease tolerance to 3-HP. That is, SEQ ID NOs:001 to 189 areincorporated into this section, SEQ ID NOs:190 to 603 are provided asnucleic acid sequences (gene, DNA) and encoded amino acid sequences(proteins) of the E. coli 3HPTGC, and SEQ ID NOs:604 to 766 are providedas sequences of the nucleic acid sequences of the Saccharomycescerevisiae 3HPTGC.

Moreover, a particular genetic modification to increase expression ofcarbonic anhydrase (for example, E. coli's cynT SEQ ID NO:337 for DNAand SEQ ID NO:544 for protein sequences), may act in a dual functionmanner to advantageously improve both 3-HP production and 3-HPtolerance. This is particularly the case when malonyl-CoA reductase isprovided for 3-HP production. FIG. 1 depicts a production pathway frommalonyl-CoA to 3-HP comprising a bi-functional malonyl-CoA reductase,and other enzymatic conversions and pathways described herein. Carbonicanhydrase is not meant to be limiting. For instance, in E. coli acarbonic anhydrase 2 is known, variously designated as can and yadF, anduse of genetic modifications in embodiments of the present invention mayuse this or other genes and their encoded enzymes. The sequences for canare provided as SEQ ID NO: 767 (EG12319 can “carbonic anhydrase 2monomer” (complement (142670..142008)) Escherichia coli K-12 substr.MG1655) and SEQ ID NO: 768 (EG12319-MONOMER carbonic anhydrase 2 monomer(complement(142670..142008)) Escherichia coli K-12 substr, MG1655).

Also, it is appreciated that genetic modifications to increase 3-HPtolerance may be further classified by genetic modifications made alongparticular respective portions of the 3HPTGC. For example, geneticmodifications may be made to polynucleotides that encode polypeptidesthat catalyze enzymatic reactions along specific portions of the of the3HPTGC and so are expected to increase production of, respectively,aromatic amino acids (tyr and phe), tryptophan (trp), ubiquinone-8,menaquinone, enterobactin, tetrahydrofolate (see respective enzymaticconversions of Group A sheet (and inputs thereto)), one or more of thepolar uncharged amino acids (gly, ser, cys, homocysteine), isoleucine,methionine (see respective enzymatic conversions of Group B sheet (andinputs thereto)), glutamine, arginine, putrescine, spermidine,aminopropylcadaverine (see see respective enzymatic conversions of GroupC sheet (and inputs thereto)), cadaverine (see respective enzymaticconversions of Group D sheet (and input thereto)), inosine-5-phosphate,xanthosine-5-phosphate, adenylo-succinate, orotidine-5′-phosphate, andany of the mono-, di-, and tri-phosphate nucleosides (i.e., adenosine,panosine, cytosine, uridine) obtainable there from (see respectiveenzymatic conversions of Group E sheet (and input thereto)), glutamate,succinate, succinate semialdehyde, oxaloacetate, and aspartate (seerespective enzymatic conversions of Group F sheet, including reactionsshown along dashed lines), such that 3-HP tolerance thereby increases asa result of such genetic modification(s). Any portion or sub-portion maybe selected for genetic modification(s) to increase 3-HP tolerance in aselected microorganism species.

As indicated, in various embodiments the combinations of geneticmodifications as described in this section are practiced in combinationwith aspects of the invention pertaining to modulation of the fatty acidsynthase system.

VIIIA. SCALES Technique

As described in WO 2010/011874, published Jan. 28, 2010, to obtaingenetic information, initial 3-HP-related fitness data was obtained byevaluation of fitness of clones from a genomic-library population usingthe SCALES technique. These clones were grown in a selective environmentimposed by elevated concentrations of 3-HP, shown to be a reliable testof 3-HP tolerance.

More particularly, to obtain data potentially useful to identify geneticelements relevant to increased 3-HP tolerance, an initial population offive representative E. coli K12 genomic libraries was produced bymethods known to those skilled in the art. The five librariesrespectively comprised 500, 1000, 2000, 4000, 8000 base pair (“bp”)inserts of E. coli K12 genetic material. Each of these libraries,essentially comprising the entire E. coli K12 genome, was respectivelytransformed into MACH1™ -T1® E. coli cells and cultured tomid-exponential phase corresponding to microaerobic conditions(OD₆₀₀˜0.2). Batch transfer times were variable and were adjusted asneeded to avoid a nutrient limited selection environment (i.e., to avoidthe cultures from entering stationary phase). Although not meant to belimiting as to alternative approaches, selection in the presence of 3-HPwas carried out over 8 serial transfer batches with a decreasinggradient of 3-HP over 60 hours. More particularly, the 3-HPconcentrations were 20 g 3-HP/L for serial batches 1 and 2, 15 g 3-HP/Lfor serial hatches 3 and 4, 10 g 3-HP/L for serial batches 5 and 6, and5 g 3-HP/L for serial batches 7 and 8. For serial batches 7 and 8 theculture media was replaced as the culture approached stationary phase toavoid nutrient limitations.

Samples were taken during and at the culmination of each batch in theselection, and were subjected to microarray analysis that identifiedsignal strengths. The individual standard laboratory methods forpreparing libraries, transformation of cell cultures, and other standardlaboratory methods used for the SCALES technique prior to array and dataanalyses are well-known in the art, such as supported by methods taughtin Sambrook and Russell, Molecular Cloning: A Laboratory Manual, ThirdEdition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (hereinafter, Sambrook and Russell, 2001). Aspectsof individual methods also are discussed in greater detail in theExamples and in the SCALES technique patent applications, U.S. PatentPublication No. 200610084098A1, tiled Sep. 20, 2005, entitled:“Mixed-Library Parallel Gene Mapping Quantitation Microarray Techniquefor Genome Wide Identification of Trait Conferring Genes” (hereinafter,the “SCALES Technique”), which is incorporated herein by reference forteaching additional details of this technique.

Microarray technology also is well-known in the art (see, e.g.<<www.affymetrix.com>>). To obtain data of which clones were moreprevalent at different exposure periods to 3-HP, Affymetrix E. coliAntisense Gene Chip arrays (Affymetrix, Santa Clara, Calif.) werehandled and scanned according to the E. coli expression protocol fromAffymetrix producing affymetrix.cel files. A strong microarray signalafter a given exposure to 3-HP indicates that the genetic sequenceintroduced by the plasmid comprising this genetic sequence confers 3-HPtolerance. These clones can be identified by numerous microarrayanalyses known in the art.

Also, for the purposes of incorporation by reference as applied in theUnited States, “A genomics approach to improve the analysis and designof strain selections,” T. E. Warnecke et al., Metabolic Engineering10(2008)154-165, is incorporated by reference herein for its additionalspecific teachings that demonstrate that SCALEs fitness data correlateswith and can be used as a surrogate of increased tolerance to 3-HP. Thisconclusion is based on the standard use of a receiver operatorcharacteristic curve (ROC) curve. ROC analysis is routinely used in themedical diagnostic field to evaluate the correlation for a diagnostictest to the actual presence or absence of a disease. Currentlydiagnostic tests used through the world in medical applications thatperform well in a ROC analysis are routinely used to identify theabsence or presence of a disease. This analysis was adapted to evaluatethe sensitivity and specificity of different microbial growth basedselections resulting in fitness values as reliable tests for 3-HPtolerance. In particular a growth based selection using serial batchcultures with decreasing levels of 3-HP was identified as a sensitiveand specific test for 3-HP tolerance. As a result clones in thisselection with a fitness metric greater than a cutoff of 0 areidentified as clones conferring tolerance to 3-HP.

The following table lists some of the genes (introduced vectors of thelibraries) that were shown have elevated fitness values, shown herein toconfer tolerance to 3-HP.

TABLE 3 SCALES Fitness Data Cumulative Cumulative Cumulative GeneFitness Gene Fitness Gene Fitness aceE 11.2 cysM 26.63 ilvC 2.61 aceF8.39 eno 6.98 ilvD 1.6 ackA 2.36 entA 1.58 ilvE 0.94 acnA 3.58 entB 0.93ilvH 1.18 acnB 3.18 entC 1.26 ilvI 1.77 adhE 3.68 entD 1 ilvM 1.02 adiA1.95 entE 1.03 ilvN 1.53 adk 2.18 entF 1.03 kbl 3.11 aldA 1.83 fbaA 2.87itaE 1.14 argA 3.94 fbaB 2.28 lysC 1.97 argB 8.94 folA 15.07 malY 2.58argC 4.02 folB 0.57 menA 3.2 argD 2.87 folC 1.72 menB 0.86 argE 2.15folD 8.54 menC 0.92 argF 2.04 folE 1.08 menD 2.33 argG 2.62 folK 1.73menE 3.06 argH 8.06 folP 2.45 menF 3.09 argI 4.06 fumA 3.84 metA 1.56aroA 2.31 fumB 2.51 metB 1.83 aroB 8.68 fumC 1.86 metC 6.08 aroC 1.95gabD 1.83 metE 2.46 aroD 1.93 gabT 1.41 metH 2.44 aroE 8.44 gapA 3.03metK 3.35 aroF 6.24 gcvH 5.9 metL 2.97 aroG 2.26 gcvP 7.91 mhpF 1.44aroH 1.61 gcvT 1.78 ndk 1.66 aroK 4 gdhA 2.84 nrdA 2.01 aroL 1.63 gldA2.08 nrdB 1.81 asd 2.96 glk 1.17 nrdD 2.79 aspC 2.82 glnA 1.34 nrdE 1.91astC 2.29 gltA 6.37 nrdF 1.25 carA 0.89 glyA 5.06 pabA 2.33 carB 1.17gmk 1.86 pabB 1.92 cynS 4.83 gnd 1.69 thrA 2.79 cysE 1.19 gpmA 2.01 thrB0.96 cysK 2.41 guaA 3.65 thrC 1.51 pabC 1.75 guaB 2.63 pheA 6.7 pfkA1.78 ilvA 12.21 pta 2.7 pflB 2.83 ilvB 2.7 purA 5.1 purB 3.65 rpiA 1.85trpC 1.56 purC 1.78 sdaA 1.62 trpD 2.48 purD 1.32 sdaB 1.22 trpE 2.85purE 1.82 serA 3.11 tynA 2.36 purF 2.04 serB 2.46 tyrA 9.1 purH 1.66serC 2.15 tyrB 1.49 purK 2.65 speA 2.09 ubiA 1.51 purL 4.83 speB 1.66ubiB 2.09 purM 3.13 speC 1.52 ubiC 2.4 purN 2.94 speD 3.43 ubiD 0.91purT 3.73 talA 1.24 ubiE 1.02 puuE 1.53 talB 4.78 ubiF 1.78 pyrB 6.36tdcB 1.87 ubiG 3.17 pyrC 14.48 tdcD 1.64 ubiH 5.35 pyrD 2.26 tdcE 1.16ubiX 1.72 pyrE 1.03 tdh 1.38 ydcW 0.89 pyrF 1.38 tktA 1.89 ydiB 0.87pyrG 2.23 tktB 1.21 ygjG 2.51 pyrH 1.78 trpA 2.45 yneI/sad 4.18 pyrI0.83 trpB 1.93 rpe 2.06

VIIIB. Analysis of the SCALES Technique

Also as described in WO 2010/011874, published Jan. 28, 2010, analysisof the 3-HP tolerance SCALEs data has led to an understanding ofinterrelationships among various identified pathways and portionsthereof. It is noted that the 3HPTGC, in its entirety, was deduced frominterrelationships between genes having elevated fitness values. Notevery enzyme of the 3HPTGC was shown in the SCALES data to have positivefitness values. This may be attributed to certain deficiencies in thecommercial arrays used to obtain that SCALES data. Accordingly, somemembers of the E. coli 3HPTGC not so derived from the SCALES geneticelement data were deduced to fill in the 3HPTGC. However, it is notedthat most of the enzymes in the 3HPTGC do have positive fitness values,and the overall fitness data in combination with the supplements andgenetic modifications data, provided herein, prove the validity of thededuction and the overall significance of the 3HPTGC being related to3-HP tolerance.

As described herein, the 3HPTGC is divided into an “upper section”comprising the glycolysis pathway, the tricarboxylic acid cycle, theglyoxylate pathway, and a portion of the pentose phosphate pathway, anda “lower section” comprising all or portions of the chorismatesuper-pathway, the carbamoyl-phosphate to carbamate pathway, thethreonine/homocysteine super-pathway the nucleotide synthesis pathway,and the polyamine synthesis pathway.

In various embodiments microorganisms are genetically modified to affectone more enzymatic activities of the 3HPTGC so that an elevatedtolerance to 3-HP may be achieved, such as in industrial systemscomprising microbial 3-HP biosynthetic activity. Also, geneticmodifications may be made to provide and/or improve one or more 3-HPbiosynthesis pathways in microorganisms comprising one or more geneticmodifications for the 3-HP toleragenic complex, thus providing forincreased 3-HP production. These latter recombinant microorganisms maybe referred to as 3-HP-syntha-toleragenic recombinant microorganisms(“3HPSATG” recombinant microorganisms).

The 3HPTGC for E. coli is disclosed in 9A, sheets 1-7 FIGS. 9B-9G (aguide for positioning these sheets to view the entire depicted 3HPTGC isprovided in FIG. 9A). As may be observed in FIGS. 9B-9G, the 3HPTGCcomprises all or various indicated portions of the following: thechorismate super-pathway, the carbamoyl-phosphate to carbamate pathway,the threonine/homocysteine super-pathway; a portion of the pentosephosphate pathway; the nucleotide synthesis pathway; theglycolysis/tricarboxylic acid cycle/glyoxylate bypass super-pathway; andthe polyamine synthesis pathway. It is noted that the chorismate pathwayand the threonine pathway are identified as super-pathways since theyrespectively encompass a number of smaller known pathways. However, theentire 3HPTGC comprises these as well as other pathways, or portionsthereof, that normally are not associated with either the chorismatesuper-pathway or the threonine/homocysteine super-pathway.

More particularly, FIG. 9A, is subdivided into the lower section, whichis further subdivided into Groups A-F and the upper section, identifiedsimply as Group F. The lower section groups are identified as follows:Group A, or “chorismate,” comprising the indicated, major portion of thechorismate super-pathway (FIGS. 9C, J, Q and X); Group B, or“threonine/homocysteine,” comprising the indicated portion of thethreonine/homocysteine pathway (FIGS. 9G, N, U and BB); Group C, or“polyamine synthesis,” comprising the indicated portion of the polyaminepathway, which includes arginine synthesis steps and also thecarbamoyl-phosphate to carbamate pathway (FIGS. 9E, L, S and Z); GroupD, or “lysine synthesis,” comprising the indicated portion of the lysinesynthesis pathway (FIGS. 9F, M, T and AA); Group F, or “nucleotidesynthesis,” comprising the indicated portions of nucleotide synthesispathways (FIGS. 9D, K, Y and H). Group F (FIGS. 9B, I, P and W)comprises the upper section of the 3HPTGC and includes the glycolysispathway, the tricarboxylic acid cycle, and the glyoxylate bypasspathway, and the indicated portions of the pentose phosphate pathway.

It is noted that particular genes are identified at enzymatic conversionsteps of the 3HPTGC in FIGS. 9B-9G. These genes are for E. coli strainK12, substrain MG1655; nucleic acid and corresponding amino acidsequences of these are available at<<http://www.ncbi.nlm.nih.gov/sites/entrez>>, and alternatively at<<www.ecocyc.org>>. As is known to one skilled in the art, some genesmay be found on a chromosome within an operon, under the control of asingle promoter, or by other interrelationships. When a nucleic acidsequence herein is referred to as a combination, such as sucC or cynTSby this is meant that the nucleic acid sequence comprises, respectively,both sucC and sucD, and both cynT and cynS. Additional control and othergenetic elements may also be in such nucleic acid sequences, which maybe collectively referred to as “genetic elements” when added in agenetic modification, and which is intended to include a geneticmodification that adds a single gene.

However, similarly functioning genes are readily found in differentspecies and strains, encoding enzymes having the same function as shownin FIGS. 9B-9G, and such genes, and the 3HPTGCs of such other speciesand strains may be utilized in the practice of the invention. This canbe achieved by the following methods, which are not meant to helimiting.

For the set of genes within the 3HPTGC of E. coli, protein sequenceswere obtained from NCBI. To identify similarly functioning genes in S.cerevisiae, a pathway comparison tool at <<www.biocyc.org>> was utilizedusing the genes identified in the E. coli 3HPTGC. For B. subtilis, thisannotated approach as used in part, and enzymes or pathway portions notobtained by that approach were obtained by a homology comparisonapproach. For the homology approach, a local blast(<<www.ncbi.nlm.nih.gov/Tools/>>) (blastp) comparison using the selectedset of E. coli proteins and Bacillus protein sequence (4096 sequences)was performed using different thresholds(<<www.ncbi.nlm.nih.gov/genomes/lproks.cgi>>). Using the homologyinformation (homology matches having E⁻¹⁰ or less E-value) the remaininggenes and enzymes were identified for the 3HPTGC for Bacillus subtilis.

Also, the latter homology approach was used for Cupriavidus necator, thefollowing table provides some examples of the homology relationships forgenetic elements of C. necator that have a demonstrated homology to E.coli genes that encode enzymes known to catalyze enzymatic conversionsteps of the 3HPTGC. This is based on the criterion of the homologoussequences having an E-value less than E⁻¹⁰. The table provides only afew of the many homologies (over 850) obtained by the comparison. Notall of the homologous sequences in C. necator are expected to encode adesired enzyme suitable for an enzymatic conversion step of the 3HPTGCfor C. necator. However, through one or more of a combination ofselection of genetic elements known to encode desired enzymaticreactions, the most relevant genetic elements are selected for the3HPTGC for this species.

TABLE 4 Homology Relationships for Genetic Elements of C. necator E.coli C. necator Gene E. coli enzyme Gene C. necator C. necator GeneSymbol E. coli enzyme product substrate Symbol E-value Product aceePyruvate acetyl-coA aceE 0 pyruvate dehydrogenase subunit E1 aceePyruvate acetyl-coA aceE 0 pyruvate dehydrogenase subunit E1 aceePyruvate acetyl-coA aceE 0 2-oxoacid dehydrogenase subunit E1 acefgi|16128108|ref|NP_414657.1| pyruvate pdhB 2.00E−102 dihydrolipoamideacetyltransferase acef gi|16128108|ref|NP_414657.1| pyruvate pdhB2.00E−25 dihydrolipoamide acetyltransferase acef Pyruvate acetyl-coApdhB 2.00E−22 dihydrolipoamide acetyltransferase acef Pyruvateacetyl-coA pdhB 1.00E−10 dihydrolipoamide acetyltransferase acefPyruvate acetyl-coA pdhL 6.00E−11 dihydrolipoamide dehydrogenase (E3)component of pyruvate dehydrogenase acef Pyruvate acetyl-coA pdhL2.00E−09 dihydrolipoamide dehydrogenase (E3) component of pyruvatedehydrogenase acef Pyruvate acetyl-coA pdhL 8.00E−08 dihydrolipoamidedehydrogenase (E3) component of pyruvate dehydrogenase acef Pyruvateacetyl-coA odhB 9.00E−36 dihydrolipoamide acetyltransferase acefPyruvate acetyl-coA bkdB 1.00E−30 branched-chain alpha- keto aciddehydrogenase subunit E2 acef pyruvate acetyl-coA bkdB 1.00E−07branched-chain alpha- keto acid dehydrogenase subunit E2 acef Pyruvateacetyl-coA bkdB 2.00E−07 branched-chain alpha- keto acid dehydrogenasesubunit E2 acna gi|16129237|ref|NP_415792.1| citrate leuC1 2.00E−19isopropylmalate isomerase large subunit acnagi|16129237|ref|NP_415792.1| citrate leuC2 7.00E−22 isopropylmalateisomerase large subunit acna gi|16129237|ref|NP_415792.1| citrate acnM 0aconitate hydratase acna gi|16129237|ref|NP_415792.1| citrate leuC36.00E−20 isopropylmalate isomerase large subunit acna Citratecis-aconitate acnA 0 aconitate hydratase acna Citrate cis-aconitateleuC4 6.00E−14 3-isopropylmalate dehydratase large subunit acna Citratecis-aconitate leuC5 1.00E−12 isopropylmalate isomerase large subunit . .. ytjc gi|16132212|ref|NP_418812.1| 3- pgam2 3.00E−25 phosphoglyceratephosphoglycerate mutase 2 protein ytjc 3-phosphoglycerate 2- pgam23.00E−25 phosphoglycerate phosphoglycerate mutase 2 protein zwfgi|16129805|ref|NP_416366.1| glucose-6- zwf1 2.00E−132glucose-6-phosphate phosphate 1-dehydrogenase zwf glucose-6-phosphateglucono- zwf2 7.00E−126 glucose-6-phosphate lactone-6- 1-dehydrogenasephosphate zwf glucose-6-phosphate glucono- zwf3 8.00E−130glucose-6-phosphate lactone-6- 1-dehydrogenase phosphate

FIGS. 9I-9N show the 3HPTGC for Bacillus subtilis, FIGS. 9P-9U show the3HPTGC for the yeast Saccharomyces cerevisiae and FIGS. 9W-9BB show the3HPTGC for Cupriavidus necator. Enzyme names for the latter are shown,along with an indication of the quantity of homologous sequences meetingthe criterion of having an E-value less than when compared against an E.coli enzyme known to catalyze a desired 3HPTGC enzymatic conversionstep.

Based on either of the above approaches, and the present existence of orrelative ease and low cost of obtaining genomic information of a givenmicroorganism species, one or both of the above approaches may beemployed to identify relevant genes and enzymes in a selectedmicroorganism species (for which its genomic sequence is known or hasbeen obtained), evaluate the relative improvements in 3-HP tolerance ofselected genetic modifications of such homologously matched andidentified genes, and thereby produce a recombinant selectedmicroorganism comprising improved tolerance to 3-HP.

Additionally, it is appreciated that alternative pathways in variousmicroorganisms may yield products of the 3HPTGC, the increasedproduction or presence of which are demonstrated herein to result inincreased 3-HP tolerance. For example, in yeast species there arealternative pathways to lysine, a product within Group D. Accordingly,alterations of such alternative pathways are within the scope of theinvention for such microorganism species otherwise falling within thescope of the relevant claim(s). Thus, in various embodiments ateinvention is not limited to the specific pathways depicted in FIGS.9A-D. That is, various pathways, and enzymes thereof, that yield theproducts shown in FIGS. 9A-D may be considered within the scope of theinvention.

It is noted that when two or more genes are shown for a particularenzymatic conversion step, these may be components of a singlemulti-enzyme complex, or may represent alternative enzymes that havedifferent control factors that control them, or are induced differently.Also, as is clear to one skilled in the art, the major reactants (i.e.,substrates) and products are shown for the enzymatic conversion steps.This is to minimize details on an already-crowded figure. For example,electron carriers and energy transfer molecules, such as NAD(P)(H) andADP/ATP, are not shown, and these (and other small-molecule reactantsnot shown in the 3HPTGC figures) are not considered “products” of the3HPTGC as that term. is used herein. Also, for at least two steps(dihydroneopterin phosphate to 7,8-dihydro-D-neopterin and1,4-dihydroxy-2-naplithoyl-CoA to 1,4-dihydroxy-2-naphthoate) no enzymeis shown because no enzyme has been known to he identified for this stepat the time of filing. Accordingly, in some embodiments the 3HPTGC isunderstood and/or taken to exclude enzymes, nucleic acid sequences, andthe like, for these steps. Also, as discussed herein, also includedwithin the scope of the invention are nucleic acid sequence variantsencoding identified enzymatic functional variants of any of the enzymesof the 3HPTGC or a related complex or portion thereof as set forthherein, and their use in constructs, methods, and systems claimedherein.

Some fitness data provided in Table 3 is not represented in the figuresof the 3HPTGC but nonetheless is considered to support geneticmodification(s) and; or supplementation to improve 3-HP tolerance. Forexample, the relatively elevated fitness scores for gcvH, gcvP and gcvT,related to the glycine cleavage system. These enzymes are involved inthe glycine/5,10-methylene-tetrahydrofolate (“5,10mTHF”) conversionpathway, depicted in FIG. 10. In the direction shown in FIG. 10, thethree enzymatically catalyzed reactions result in decarboxylation ofglycine (a 3HPTGC product, see 9D), production of 5,10-methylene-THFfrom tetrahydrofolate (“THF”), and production of NADH from NAD⁺. The5,10-methylene-THF product of this complex is a reactant inenzymatically catalyzed reactions that are part of the following: folatepolyglutamylation; panthothenate biosynthesis; formylTHF biosynthesis;and de novo biosynthesis of pyrimidine deoxyribonucleotides. Overall,genetic modifications in a microorganism directed to the enzymes, andenzymatic catalytic steps thereof, shown in Table 3 but not representedin FIGS. 9B-G are considered part of the invention (as are theirfunctional equivalents for other species), wherein such geneticmodifications result in an increase in 3-HP tolerance.

VIIC. Genetic Modifications and Supplementations of the 3HPTGC

For various embodiments of the invention the genetic modifications toany pathways and pathway portions of the 3HPTGC and arty of the 3-HPbio-production pathways may be described to include various geneticmanipulations, including those directed to change regulation of, andtherefore ultimate activity of, a enzyme, or enzymatic activity of anenzyme identified in any of the respective pathways. Such geneticmodifications may be directed to transcriptional, translational, andpost translational modifications that result in a change of enzymeactivity and/or overall enzymatic conversion rate under selected and/oridentified culture conditions, and/or to provision of additional nucleicacid sequences (as provided in some of the Examples) so as to increasecopy number and/or mutants of an enzyme of the 3HPTGC.

Specific methodologies and approaches to achieve such geneticmodification are well known to one skilled in the art, and include, butare not limited to: increasing expression of an endogenous geneticelement; decreasing functionality of a repressor gene; introducing, aheterologous genetic element; increasing copy number of a nucleic acidsequence encoding a polypeptide catalyzing an enzymatic conversion stepof the 3HPTGC; mutating a genetic element to provide a mutated proteinto increase specific enzymatic activity; over-expressing;under-expressing; over-expressing a chaperone; knocking out a protease;altering or modifying feedback inhibition; providing an enzyme variantcomprising one or more of an impaired binding site for a repressorand/or competitive inhibitor; knocking out a repressor gene; evolution,selection and/or other approaches to improve mRNA stability. Randommutagenesis may be practiced to provide genetic modifications of the3HPTGC that may fall into any of these or other stated approaches. Thegenetic modifications further broadly fall into additions (includinginsertions), deletions (such as by a nutation) and substitutions of oneor more nucleic acids in a nucleic acid of interest. In variousembodiments a genetic modification results in improved enzymaticspecific activity and/or turnover number of an enzyme. Without beinglimited, changes may be measured by one or more of the following: K_(M); K_(cat); and K_(avidity).

Such genetic modifications overall are directed to increase enzymaticconversion at at least one enzymatic conversion step of the 3HPTGC so asto increase 3-HP tolerance of a microorganism so modified. Also, theenzymatic conversion steps shown in FIGS. 9A-D may be catalyzed byenzymes that are readily identified by one skilled in the art, such asby searching for the enzyme name corresponding to the gene name at aparticular enzymatic conversion step in FIGS. 9A-D, and then identifyingenzymes, such as in other species, having the same name and function.The latter would be able to convert the respective reactant(s) to therespective product(s) for that enzymatic conversion step. Publicdatabase sites, such as <<www.metacyc.org>>, <<www.ecocyc.org>>,<<www.biocyc.org>>, and <<www.nchi.gov>>, have associated tools toidentify such analogous enzymes.

Also, although the MIC analysis is used frequently herein as an endpointto indicate differences in microorganism growth when placed in various3-HP concentrations for a specified time, this is by no means consideredto be the only suitable metric to determine a difference, such as animprovement, in microorganism tolerance based on aspects of theinvention. Without being limiting, other suitable measurement approachesmay include growth rate determination, lag time determination, changesin optical density of cultures at specified culture durations, number ofdoublings of a population in a given time period and, for microorganismsthat comprise 3-HP production capability, overall 3-HP production in aculture system in which 3-HP accumulates to a level inhibitory to acontrol microorganism lacking genetic modifications that increaseenzymatic conversion at one or more enzymatic conversion steps of the3HPTGC. This may result in increased productivities, yields or titers.

It is generally appreciated that a useful metric to assess increases in3-HP tolerance can be related to a microorganism's or a microorganismculture's ability to grow while exposed to 3-HP over a specified periodof time. This can be determined by various quantitative and/orqualitative analyses and endpoints, particularly by comparison to anappropriate control that lacks the 3-HP tolerance-related geneticmodification(s) and/or supplements as disclosed and discussed herein.Time periods for such assessments may be, but are not limited to: 12hours; 24 hours; 48 hours; 72 hours; 96 hours; and periods exceeding 96hours. Varying exposure concentrations of 3-HP may be assessed to moreclearly identify a 3-HP tolerance improvement. The following paragraphsprovide non-limiting examples of approaches that may be used todemonstrate differences in a microorganism's ability to grow and/orsurvive in the presence of 3-HP in its culture system when teachings ofthe present invention are applied to the microorganism and/or theculture system.

FIGS. 15A-O provide data from various control microorganism responses todifferent 3-HP concentrations. The data in these figures is shownvariously as changes maximum growth rate (μ_(max)), changes in opticaldensity (“OD”), and relative doubling, times over a given period, here24 hours.

Determination of growth rates, lag times and maximum growth rates arecommonly used analyses to develop comparative metrics. FIGS. 15A, 15D,15G, 15J, and 15M demonstrate changes in maximum growth rates over a24-hour test period for the indicated species under the indicatedaerobic or anaerobic test conditions. When representing this data for arange of concentrations of a chemical of interest that is believed toxicand/or inhibitory to growth, this representation is termed a “toleragam”herein. Here, growth toleragrams are generated by measuring the specificgrowth rates of microorganisms subjected to growth conditions including,varying amounts of 3-HP.

Further, FIG. 15P compares the growth toleragrams of a controlmicroorganism culture with a microorganism in which genetic modificationwas made to increase expression of cynTS (in Group C of the 3HPTGC). Thecurve for a cynTS genetic modification in E. coli shows increasingmaximum growth rate with increasing 3-HP concentration over a 24-hourevaluation period for each 3-HP concentration. This provides aqualitative visually observable difference. However, the greater areaunder the curve for the cynTS genetic modification affords aquantitative difference as well, which may be used for comparativepurposes with other genetic modifications intended to improve 3-HPtolerance. Evaluation of such curves may lead to more effectiveidentification of genetic modifications and/or supplements, andcombinations thereof.

FIGS. 15B, 15E, 15F, 15K, and 15N demonstrate a control microorganismresponses to different 3-HP concentrations wherein optical density(“OD,” measured at 600 nanometers; at 24 hours is the metric used. OD600is a conventional measure of cell density in a microorganism culture.For E. coli under aerobic condition, FIG. 15B demonstrates a dramaticreduction in cell density at 24 hours starting at 30 g/L3-HP. FIG. 15Dshows a relatively sharper and earlier drop for E. coli under anaerobicconditions.

FIGS. 15C, 15F, 15I, 15E, and 15O demonstrate control microorganismresponses to different 3-HP concentrations wherein the number of celldoublings during the 24-hour period are displayed.

The above is intended as a non-limiting description of various ways toassess 3-HP tolerance improvements. Generally, demonstrable improvementsin growth and/or survival are viewed as ways to assess an increase intolerance, such as to 3-HP.

Embodiments of the present invention may result from introduction of anexpression vector into a host microorganism, wherein the expressionvector contains a nucleic acid sequence coding for an enzyme that is, oris not, normally found in a host microorganism. With reference to thehost microorganism's genome prior to the introduction of theheterologous nucleic acid sequence, then, the nucleic acid sequence thatcodes for the enzyme is heterologous (whether or not the heterologousnucleic acid sequence is introduced into that genome).

Generally, it is within the scope of the invention to provide one ormore genetic modifications to increase a recombinant microorganism'stolerance to 3-HP by any one or more of the approaches described herein.Thus, within the scope of arty of the above-described alternatives andembodiments thereof are the composition results of respective methods,that is, genetically modified microorganisms that comprise the one ormore, two or more, three or more, etc. genetic modifications referred totoward obtaining increased tolerance to 3-HP.

Also, it is within the scope of the invention to provide, in a suitableculture vessel comprising a selected microorganism, one or moresupplements that are intermediates or end products (collectively,“products”) of the 3HPTGC. Table 5 recites a non-limiting listing ofsupplements that tray be added in a culture vessel comprising agenetically modified microorganism comprising one or more geneticmodifications to the 3HPTGC and/or 3-HP production pathways. Forexample, not to be limiting, one or more of lysine, methionine, andbicarbonate may be provided. Such supplement additions may be combinedwith genetic modifications, as described herein, of the selectedmicroorganism.

TABLE 5 TGC Concentration, Supplement Source Group g/L Note TyrosineSigma, St. Louis, MO A 0.036 dissolve in 0.01 KOH, pH final to 7Phenylalanine Sigma, St. Louis, MO A 0.0664 Tryptophan Sigma, St. Louis,MO A 0.0208 Shikimate Sigma, St. Louis, MO A 0.1 p-aminobenzoate MPBiomedicals, Aurora, A 0.069 OH Dihydroxybenzoate Sigma, St. Louis, MO A0.077 Tetrahydrofolate Sigma, St. Louis, MO A 0.015 10% DMSOHomocysteine MP Biomedicals, Aurora, B 0.008 OH Isoleucine Sigma, St.Louis, MO B 0.0052 Serine Sigma, St. Louis, MO B 1.05 Glycine FisherScientific, Fair B 0.06 Lawn, NJ Methionine Sigma, St. Louis, MO B 0.03Threonine Sigma, St. Louis, MO B 0.0476 2-oxobutyrate Fluka Biochemika,B 0.051 Hungary Homoserine Acros Organics, NJ B 0.008 Aspartate Sigma,St. Louis, MO B 0.0684 Putrescine MP Biomedicals, Salon, C 0.9 OHCadaverine MP Biomedicals, Salon, O C 0.6 Spermidine MP Biomedicals,Salon, C 0.5 OH Ornithine Sigma, St. Louis, MO C 0.2 Citrulline Sigma,St. Louis, MO C 0.2 Bicarbonate Fisher Scientific, Fair C 1 Lawn, NJGlutamine Sigma, St. Louis, MO C 0.09 dissolve in 1M HCl, pH final to 7Lysine Sigma, St. Louis, MO D 0.0732 Uracil Sigma, St. Louis, MO E 0.224Citrate Fisher Scientific, Fair F 2 Lawn, NJ Chorismate Group Mix Seeabove A See respective (includes all Group A concentrations supplementslisted above above) Homocysteine Group See above B See respective Mix(includes all Group concentrations B supplements listed above above)Polyamine Group Mix See above C See respective (includes all Group Cconcentrations supplements listed above above)

Further as to supplements, as to Group C regarding polyamine synthesis,the results of the examples demonstrate that 3-HP tolerance of E. coliwas increased by adding the polyamines putrescine, spermidine andcadaverine to the media. Minimum inhibitory concentrations (MICs) for E.coli K12 in control and supplemented media were as follows: in M9minimal media supplemented with putrescine 40 g/L, in M9 minimal mediasupplemented with spermidine 40 g/L, in M9 minimal media supplementedwith cadaverine 30 g/L. Minimum inhibitory concentrations (MICs) foradded sodium bicarbonate in M9 minimal media was 30 L. The Minimuminhibitory concentrations (MICs) for E. coli K12 in 100 g/L stocksolution 3-HP was 20 g/L.

Further, in view of the increase over the control MIC with sodiumbicarbonate supplementation, other alteration, such as regulation and/orgenetic modification of carbonic anhydrase, such as providing aheterologous nucleic acid sequence to a cell of interest, where thatnucleic acid sequence encodes a polypeptide possessing carbonicanhydrase activity are considered of value to increase tolerance to 3-HP(such as in combination with other alterations of the 3HPTGC).Similarly, and as supported by other data provided herein, alterationsof the enzymatic activities, such as by genetic modification(s) ofenzymers) along the 3HPTGC pathway portions that lead to arginine,putrescine, cadaverine and spermidine, are considered of value toincrease tolerance to 3-HP (such as in combination with otheralterations of the 3HPTGC).

It is appreciated that the results of supplementations evaluationsprovide evidence of the utility of direct supplementation into a culturemedia, and also of improving 3-HP tolerance by a genetic modificationroute, such as is provided in some examples herein. It is appreciatedthat increasing the concentration of a product of a 3HPTGC enzymaticconversion step, such as by a genetic modification, whether bysupplementation and/or genetic modification(s), may be effective toincrease the intracellular concentration of one or more 3HPTGC productsin a microorganism and/or in the media in which such microorganism iscultured.

Taken together, the fitness data and subsequently obtained data from theexamples related to genetic modifications and/or supplements pertainingto the 3HPTGC support a concept of a functional relationship betweensuch alterations to increase enzymatic conversion along the pathways ofthe 3HPTGC and the resulting functional increase in 3-HP tolerance in amicroorganism cell or culture system. This is observable for the 3HPTGCas a whole and also within and among its defined groups.

Further, tables 47, 48, 50, 52, 53, and 56, incorporated into thissection, provide non-limiting examples supplements additions, geneticmodifications, and combinations of supplements additions and geneticmodifications. Additional supplementations, genetic modifications, andcombinations thereof, may be made in view of these examples and thedescribed methods of identifying genetic modifications toward achievingan elevated tolerance to 3-HP in a microorganism of interest. Particularcombinations may involve only the 3HPTGC lower section, includingcombinations involving two or more, three or more, or four or more, ofthe five groups therein (each involving, supplement additions and/orgenetic modification), any of these in various embodiments alsocomprising one or more genetic modifications or supplement additionsregarding the 3HPTGC upper section. Subject matter in the Examples isincorporated into this section to the extent not already present.

Based on these results, it is appreciated that in various embodiments ofthe invention, whether methods or compositions, as a result of geneticmodification and/or supplementation of reactants of the 3HPTGC, thealteration(s) directed to the 3HPTGC are effective to increase 3-HPtolerance by at least 5 percent, at least 10 percent, at least 20percent, at least 30 percent, or at least 50 percent above a 3-HPtolerance of a control microorganism, lacking said at least one 3HPTGCgenetic modification.

As is appreciated by the examples, any of the genetically modifiedmicroorganisms of the invention may be provided in a culture system andutilized, such as for the production of 3-HP. In some embodiments, oneor more supplements (that are products of the 3HPTGC enzymaticconversion steps) are provided to a culture system to further increaseoverall 3-HP tolerance in such culture system.

Increased tolerance to 3-HP, whether of a microorganism or a culturesystem, may be assessed by any method or approach known to those skilledin the art, including but not limited to those described herein.

The genetic modification of the 3HPTGC upper portion may involve any ofthe enzymatic conversion steps. One, non-limiting example regards thecarboxylic acid cycle. It is known that the presence and activity of theenzyme citrate synthase (E.C. 2.3.3.1 (previously 4.1.3.7)), whichcatalyzes the first step in that cycle, controls the rate of the overallcycle (i.e., is a rate-limiter). Accordingly, genetic modification of amicroorganism, such as to increase copy numbers and/or specificactivity, and/or other related characteristics (such as lower effect ofa feedback inhibitor or other control molecule), may include amodification of citrase synthase. Ways to effectuate such change forcitrate synthase may utilize any number of laboratory techniques, suchas are known in the art, including approaches described herein for otherenzymatic conversion steps of the 3HPTGC. Further, several commonlyknown techniques are described in U.S. Pat. Nos. 6,110,714 and7,247,459, both assigned to Ajinomoto Co., Inc., both of which areherewith incorporated by reference for their respective teachings aboutamplifying citrate synthase activity (specifically, cols. 3 and 4, andExamples 3 and 4, of U.S. Pat. No. 6,110,714, and cols. 11 and 12(specifically Examples (1) and (2)) of U.S. Pat. No. 7,247,459).

In various embodiments E. coli strains are provided that compriseselected gene deletions directed to increase enzymatic conversion in the3HPTGC and accordingly increase microorganism tolerance to 3-HP. Forexample, the following genes, which are associated with repression ofpathways in the indicated 3HPTGC Groups, may be deleted: Group A—tyrR,trpR; Group B—motJ; Group C—purR; Group D—lysR; Group E—nrdR. There arefor E. coli and it is known and determinable by one skilled in the artto identify and genetically modify equivalent repressor genes in thisand other species.

A disruption of gene function may also be effectuated, in which thenormal encoding of a functional enzyme by a nucleic acid sequence hasbeen altered so that the production of the functional enzyme in amicroorganism cell has been reduced or eliminated. A disruption maybroadly include a gene deletion, and also includes, but is not limitedto gene modification (e.g., introduction of stop codons, frame shiftmutations, introduction or removal of portions of the gene, introductionof a degradation signal), affecting mRNA transcription levels and/orstability, and altering the promoter or repressor upstream of the geneencoding the polypeptide. In some embodiments, a gene disruption istaken to mean any genetic modification to the DNA, mRNA encoded from theDNA, and the amino acid sequence that results in at least a 50 percentreduction of enzyme function of the encoded gene in the microorganismcell.

Further, as to the full scope of the invention and for variousembodiments, it is recognized that the above discussion and the examplesare meant to be exemplary and not limiting. Genetic manipulations may bemade to achieve a desired alteration in overall enzyme function, such asby reduction of feedback inhibition and other facets of control,including alterations in DNA transcriptional and RNA translationalcontrol mechanisms, improved mRNA stability, as well as use of plasmidshaving an effective copy number and promoters to achieve an effectivelevel of improvement. Such genetic modifications may be chosen and/orselected for to achieve a higher flux rate through certain basicpathways within the 3HPTGC and so may affect general cellular metabolismin fundamental and/or major ways. Accordingly, in certain alternativesgenetic modifications are made more selectively, to other parts of the3HPTGC.

Further, based on analysis of location and properties of committedsteps, feedback inhibition, and other factors and constraints, invarious embodiments at least one genetic modification is made toincrease overall enzymatic conversion for one of the following enzymesof the 3HPTGC: 2-dehydro-3-deoxyphosphoheptonate aldolase (e.g., aroF,aroG, aroH); cyanase (e.g., cynS); carbonic anhydrase (e.g., cynT);cysteine synthase B (e.g., cysM); threonine deaminase (e.g., ilvA);ornithine decarboxylase (e.g., speC, speF); adenosylmethioninedecarboxylase (e.g., speD); and spermidine synthase (e.g., speE).Genetic modifications may include increasing copy numbers of nucleicacid sequences encoding these enzymes, and providing modified nucleicacid sequences that have reduced or eliminated feedback inhibition,control by regulators, increased affinity for substrate, and othermodifications. Thus, one aspect of the invention is to geneticallymodify one or more of these enzymes in a manner to increase enzymaticconversion at one or more 3HPTGC enzymatic conversion steps so as toincrease flux and/or otherwise modify reaction flows through the 3HPTGCso that 3-HP tolerance is increased. In addition to the examples whichpertain to genetic modifications regarding aroH and cyanase (withcarbonic anhydrase), respectively, the following examples are provided.It is noted that in E. coli a second carbonic anhydrase enzyme is known.This is identified variously as Can and yadf.

Also, it is appreciated that various embodiments of the invention maycomprise genetic; modifications of the 3HPTGC (as may be provided in amicroorganism, as described herein), and/or supplements thereof,excluding any one or more designated enzymatic conversion steps, productadditions, and/or specific enzymes. For example, an embodiment of theinvention may comprise genetic modifications of the 3HPTGC in amicroorganism, however excluding those of Group A, or of Groups A and B,or of a defined one or more members of the 3HPTGC (which may be anysubset of the 3HPTGC members).

For example, without being limiting, a modified 3HPTGC may comprise allmembers of the 3HPTGC as depicted herein except the degradative form ofarginine decarboxylase (adiA, which is known to be induced in richmedium at low pH under anaerobic conditions in the presence of excesssubstrate), or other subsets excluding such degradative argininedecarboxylase and other selected enzyme steps. Other modified 3HPTGCcomplexes may also he practiced in various embodiments. Based on thenoted induction of adiA, the use of the degadative form of argininedecarboxylase is not be considered within the scope of the 3HPTGC for3-HP tolerance improvement as practiced under aerobic conditions.

Moreover, various non-limiting aspects of the invention may include, butare not limited to:

A genetically modified (recombinant) microorganism comprising a nucleicacid sequence that encodes a polypeptide with at least 85% amino acidsequence identity to any of the enzymes of any of 3-HP tolerance-relatedor biosynthetic pathways, wherein the polypeptide has enzymatic activityand specificity effective to perform the enzymatic reaction of therespective 3-HP tolerance-related or biosynthetic pathway enzyme, andthe recombinant microorganism exhibits greater 3-HP tolerance and/or3-HP bio-production than an appropriate control microorganism lackingsuch nucleic acid sequence.

A genetically modified (recombinant) microorganism comprising a nucleicacid sequence that encodes a polypeptide with at least 90% amino acidsequence identity to any of the enzymes of any of 3-HP tolerance-relatedor biosynthetic pathways, wherein the polypeptide has enzymatic activityand specificity effective to perform the enzymatic reaction of therespective 3-HP tolerance related or biosynthetic pathway enzyme, andthe recombinant microorganism exhibits greater 3-HP tolerance and/or3-HP bio-production than an appropriate control microorganism lackingsuch nucleic acid sequence.

A genetically modified (recombinant) microorganism comprising a nucleicacid sequence that encodes a polypeptide with at least 95% amino acidsequence identity to any of the enzymes of any of 3-HP tolerance-relatedor biosynthetic pathways, wherein the polypeptide has enzymatic activityand specificity effective to perform the enzymatic reaction of therespective 3-HP tolerance-related or biosynthetic pathway enzyme, andthe recombinant microorganism exhibits greater 3-HP tolerance and/or3-HP bio-production than an appropriate control microorganism lackingsuch nucleic acid sequence. In some embodiments, the at least onepolypeptide has at least 99% or 100% sequence identity to at least oneof the enzymes of a 3-HPTGC pathway and/or a 3-HP biosynthetic pathway.

In one aspect of the invention the identity values in the precedingparagraphs are determined using the parameter set described above forthe FASTDB software program, or BLASTP or BLASTN, such as version 2.2.2,using default parameters. Further, for all specifically recitedsequences herein it is understood that conservatively modified variantsthereof are intended to be included within the invention. In accordancewith the present disclosure, in various embodiments the inventioncontemplates a genetically modified (e.g., recombinant) microorganismcomprising a heterologous nucleic acid sequence that encodes apolypeptide that is an identified enzymatic functional variant of any ofthe enzymes of any of 3-HP tolerance related pathways, or pathwayportions (i.e., of the 3HPTGC), or other enzyme disclosed herein (e.g.,of a 3-HP production pathway), wherein the polypeptide has enzymaticactivity and specificity effective to perform the enzymatic reaction ofthe respective 3-HP tolerance-related or other enzyme, so that therecombinant microorganism exhibits greater 3-HP tolerance or otherfunction than an appropriate control microorganism lacking such nucleicacid sequence. Relevant methods of the invention also are intended to bedirected to identified enzymatic functional variants and the nucleicacid sequences that encode them. Embodiments may also comprise otherfunctional variants.

In some embodiments, the invention contemplates a recombinantmicroorganism comprising at least one genetic modification effective toincrease 3-hydroxypropionic acid (“3-HP”) production, wherein theincreased level of 3-HP production is greater than the level of 3-HPproduction in the wild-type microorganism, and at least one geneticmodification of the 3-HP Toleragenic Complex (“3HPTGC”). In someembodiments, the wild-type microorganism produces 3-HP. In someembodiments, the wild-type microorganism does not produce 3-HP. In someembodiments, the recombinant microorganism comprises at least onevector, such as at least one plasmid, wherein the at least one vectorcomprises at least one heterologous nucleic acid molecule.

In some embodiments of the invention, the at least one geneticmodification of the 3HPTGC is effective to increase the 3-HP toleranceof the recombinant microorganism above the 3-HP tolerance of a controlmicroorganism, wherein the control microorganism lacks the at least one3HPTGC genetic modification. In some embodiments, the 3-HP tolerance ofthe recombinant microorganism is increased above the 3-HP tolerance of acontrol microorganism by about 5%, 10%, or 20%. In some embodiments, the3-HP tolerance of the recombinant microorganism is increased above the3-HP tolerance of a control microorganism by about 30%, 40%, 50%, 60%,80%, or 100%.

Also, in various embodiments, the at least one genetic modification ofthe 3HPTGC encodes at least one polypeptide exhibiting at least oneenzymatic conversion of at least one enzyme of the 3HPTGC, wherein therecombinant microorganism exhibits an increased 3-HP tolerance at leastabout 5, 10, 20, 30, 40, 50, 60, or 100 percent greater, or more, thanthe 3-HP tolerance of a control microorganism lacking the at least onegenetic modification of the 3HPTGC. Any evaluations for such toleranceimprovements may be based on a Minimum inhibitory Concentrationevaluation in a minimal media.

In some embodiments, the microorganism further comprises at least oneadditional genetic modification encoding at least one polypeptideexhibiting at least one enzymatic conversion of at least one enzyme of asecond Group different from the genetic modification of a first Group ofthe 3HPTGC, wherein the recombinant microorganism exhibits an increased3-HP tolerance at least about 5, 10, 20, 30, 40, 50, 60, or 100 percentgreater, or more, than the 3-HP tolerance of a control microorganismlacking all said genetic modifications of the 3HPTGC. In the variousembodiments, the at least one additional genetic modification furthercomprises a genetic modification from each of two or more, or three ormore, of the Groups A-F.

For example, the genetic modifications may comprise at least one geneticmodification of Group A and at least one genetic modification of GroupB, at least one genetic modification of Group A and at least one geneticmodification of Group C, at least one genetic modification of Group Aand at least one genetic modification of Group D, at least one geneticmodification of Group A and at least one genetic modification of GroupE, at least one genetic modification of Group B and at least one geneticmodification of Group C, at least one genetic modification of Group Band at least one genetic modification of Group D, at least one geneticmodification of Group B and at least one genetic modification of GroupE, at least one genetic modification of Group C and at least one geneticmodification of Group D, at least one genetic modification of Group Cand at least one genetic modification of Group E, or at least onegenetic modification of Group D and at least one genetic modification ofGroup E. Any such combinations may be further practiced with Group Fgenetic modifications.

In some embodiments, the recombinant microorganism comprises one or moregene disruptions of 3HPTGC repressor genes selected from tyrR, trpR,metJ, argR, purR, lysR and nrdR.

In some embodiments, the at least one genetic modification of the 3HPTGCcomprises means to increase expression of SEQ ID NO: 129 (Irok peptide).In some embodiments, the recombinant microorganism is an E. coli strain.In some embodiments, the recombinant microorganism is a Cupriavidusnecator strain.

In some embodiments, the at least one genetic modification encodes atleast one polypeptide with at least 85% amino acid sequence identity toat least one of the enzymes of a 3-HPTGC pathway, a 3-HP biosyntheticpathway, and/or SEQ ID NO: 129 (Irok).

Some embodiments of the invention contemplate a culture system. In someembodiments, the culture system comprises a genetically modifiedmicroorganism as described herein and a culture media. Such geneticallymodified microorganism may comprise a single genetic modification of the3HPTGC, or any of the combinations described herein, and mayadditionally comprise one or more genetic modifications of a 3-HPproduction pathway. In some embodiments, the culture media comprises atleast about 1 g/L, at least about 5 g/L, at least about 10 g/L, at leastabout 15 g/L, or at least about 20 g/L of 3-HP. In some embodiments, theculture system comprises a 3HPTGC supplement at a respectiveconcentration such as that shown herein.

In some embodiments the invention contemplates a method of making agenetically modified microorganism comprising providing at least onegenetic modification to increase the enzymatic conversion of thegenetically modified microorganism over the enzymatic conversion of acontrol microorganism, wherein the control microorganism lacks the atleast one genetic modification, at an enzymatic conversion step of the3-hydroxypropionic acid Toleragenic Complex (“3HPTGC”), wherein thegenetically modified microorganism synthesizes 3-HP. In someembodiments, the control microorganism synthesizes 3-HP. In someembodiments, the at least one genetic modification increases the 3-HPtolerance of the genetically modified microorganism above the 3-HPtolerance of the control microorganism.

In some embodiments, the 3-HP tolerance of the genetically modifiedmicroorganism is at least about 5 percent, at least about 10 percent, atleast about 20 percent, at least about 30 percent, at least about 40percent, at least about 50 percent, or at least about 100 percent abovethe 3-HP tolerance of the control microorganism. In some embodiments,the 3-HP tolerance of the genetically modified microorganism is fromabout 50 to about 300 percent above the 3-HP tolerance of the controlmicroorganism, based on a Minimum Inhibitory Concentration evaluation ina minimal media. In some embodiments, the genetically modifiedmicroorganism further comprises one or more gene disruptions of 3HPTGCrepressor genes selected from tyrR, trpR, metJ, argR, purR, lysR andnrdR. In some embodiments, the control microorganism does not synthesize3-HP. In some embodiments, providing at least one genetic modificationcomprises providing at least one vector. In some embodiments, the atleast one vector comprises at least one plasmid, in some embodiments,providing at least one genetic modification comprises providing at leastone nucleic acid molecule. In some embodiments, the at least one nucleicacid molecule is heterologous. In some embodiments, the at least onenucleic acid molecule encodes SEQ ID NO: 129 (Irok).

In some embodiments, genetic modifications are made to increaseenzymatic conversion at an enzymatic conversion step identified to havean elevated fitness score in Table 3 and/or evaluated in the Examples.Enzymes that catalyze such reactions are numerous and include cyanaseand carbonic anhydrase.

Also, it is appreciated that various embodiments of the invention may bedirected to amino acid sequences of enzymes that catalyze the enzymaticconversion steps of the 3HPTGC for any species. More particularly, theamino acid sequences of the 3HPTGC for FIGS. 9A-D are readily obtainablefrom one or more of commonly used bioinformatics databases (e.g.,<<www.ncbi.gov>>; <<www.metacyc.org>>) by entering a respective gene foran enzymatic conversion step therein.

IX. Combinations of Genetic Modifications

As described in U.S. Provisional Patent Application No. 61/246,141,incorporated by reference and to which priority is claimed, variouscombinations of genetic modifications may be implemented in variousembodiments of the invention. These are described in the followingparagraphs and Tables 6A, 6B and 7, noting that the first paragraphsrelated to various forms of malonyl-CoA reductace that may be used inthe combinations.

Various embodiments of the present invention comprise a geneticallymodified microorganism comprising at least one genetic modification tointroduce or increase malonyl-CoA-reductase enzymatic activity,including by introducing a polynucleotide that expresses a functionalequivalent of the malonyl-CoA-reductase provided herein. A functionalequivalent of malonyl-CoA-reductase enzymatic activity is capable ofincreasing enzymatic activity for conversion of malonyl-CoA to malonatesemialdehyde, malonate semialdehyde to 3-HP, or both.

In some embodiments, the amino acid sequence of themalonyl-CoA-reductase comprises SEQ ID NO:783. In other embodiments, themalonyl-CoA-reductase comprises a variant of any of SEQ NOs:783 to 791exhibiting malonyl-CoA-reductase enzymatic activity.

The amino acid sequence of the malonyl-CoA-reductase can comprise anamino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 92%,95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs:783 to 791.

In some embodiments, at least one genetic modification comprisesproviding a polynucleotide that encodes an amino acid sequencecomprising one of, or a functional portion of, any of SEQ ID NOs: 783 to791. In various embodiments, at least one genetic modification comprisesproviding a polynucleotide that encodes an amino acid sequence having atleast 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%sequence identity to any of SEQ ID NOs: 783 to 791.

In exemplary embodiments, the polynucleotide is codon-optimizedfor aselected microorganism species to encode any one of SEQ ID NOs: 783 to791. In various embodiments, the polynucleotide is codon-optimized toencode an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%,90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQNOs: 783 to 791. The polynucleoticle can be codon-optimized for E. coli,for example.

In some embodiments, the genetically modified microorganism that sopossesses malonyl-CoA-reductase genetic modification(s) additionallycomprises at least one genetic modification to increase, in thegenetically modified microorganism, a protein function selected from theprotein functions of Table 6A (Glucose transporter function (such as bygalP), pyruvate dehydrogenase Elp, dihydrolipoamide acetyltransferase,and pyruvate dehydrogenase E3). In certain embodiments, the geneticallymodified microorganism comprises at least one genetic modification toincrease two, three, or four protein functions selected from the proteinfunctions of Table 6A.

In some embodiments, such genetically modified microorganismadditionally comprises at least one genetic modification to decreaseprotein functions selected from the protein functions of Table 6B,lactate dehydrogenase, pyruvate formate lyase, pyruvate oxidase,phosphate acetyltransferase, histidyl phosphorylatable protein (of PTS),phosphoryl transfer protein (of PTS), and the polypeptide chain (ofPTS).

In various embodiments, such genetically modified microorganismcomprises at least one genetic modification to decrease enzymaticactivity of two, three, four, five, six, or seven protein functionsselected from the protein functions of Table 6B. Also, in variousembodiments at least one, or more than one, genetic modification is madeto modify the protein functions of Table 7 in accordance with theComments therein.

It will be appreciated that, in various embodiments, there can be manypossible combinations of increases in one or more protein functions ofTable 6A, with reductions in one or more protein functions of Table 6Ain the genetically modified microorganism comprising at least onegenetic modification to provide or increase malonyl-CoA-reductaseprotein function (i.e, enzymatic activity). Protein functions can beindependently varied, and any combination (i.e., a full factorial) ofgenetic modifications of protein functions in Tables 6A, 6B, and 7herein can be adjusted by the methods taught and provided into saidgenetically modified microorganism.

In some embodiments, at least one genetic modification to decreaseenzymatic activity is a gene disruption. In some embodiments, at leastone genetic modification to decrease enzymatic activity is a genedeletion.

In various embodiments, to obtain 3-hydroxypropionic acid (3-HP) as adesired product, the genetically modified microorganism comprises aprotein function effective for converting malonate semialdehyde to 3-HP.The protein function effective for converting malonate semialdehyde to3-HP can be native to the microorganism, but that is by no meansnecessary.

In some embodiments, the protein function effective for convertingmalonate semialdehyde to 3-HP is a native or mutated form of mmsB fromPseudomonas aeruginosas, or a functional equivalent thereof.Alternatively, or additionally, this protein function can be a native ormutated form of ydfG, or a functional equivalent thereof.

Certain embodiments of the invention additionally comprise a geneticmodification to increase the availability of the cofactor NADPH, whichcan increase the NADPH/NADP+ ratio as may be desired. Non-limitingexamples for such genetic modification are pgi. (E.C. 5.3.1.9, in amutated form), pntAB (E.C. 1.6.1.2), overexpressed, gapA(E.C.1.2.2.12):gapN (E.C. 1.2.1.9, from Streptococcus mutans)substitution/replacement, and disrupting or modifying a solubletranshydrogenase such as sthA. (E.C. 1.6.1.2), and/or geneticmodifications of one or more of zwf (E.C. 1.1.1.49), gnd (E.C.1.1.1.44), and edd (E.C. 4.2.1.12). Sequences of these genes areavailable at www.metacyc.org. Also, the sequences for the genes andencoded proteins for the E. coli gene names shown in Tables 6A, 6B, and7 are provided in U.S. Provisional Patent Application No. 61/246,141,incorporated herein in its entirety and for such sequences, and also areavailable at www.ncbi.gov as well as www.metacyc.org or www.ecocyc.org.

In some embodiments, the genetic modification increases microbialsynthesis of 3-HP above a rate or titer of a control microorganismlacking said at least one genetic modification to produce 3-HP. In someembodiments, the genetic modification is effective to increase enzymaticconversions to 3-HP by at least about 5 percent, at least about 10percent, at least about 20 percent, at least about 30 percent, or atleast about 50 percent above the enzymatic conversion of a controlmicroorganism lacking the genetic modification.

TABLE 6A E.C. Gene Name in Enzyme Function Classification E. coliGlucose transporter N/A galP Pyruvate dehydrogenase E1p 1.2.4.1 aceElipoate 2.3.1.12 aceF acetyltransferase/dihydrolipoamideacetyltransferase Pyruvate dehydrogenase 1.8.1.4 lpd E3 (lipoamidedehydrogenase)

TABLE 6B E.C. Gene Name in Enzyme Function Classification E. coliLactate dehdrogenase 1.1.1.28 ldhA Pyruvate formate lyase 2.3.1.— pflB(B “inactive”) Pyruvate oxidase 1.2.2.2 poxB Phosphate acetyltransferase2.3.1.8 Pta Heat stable, histidyl N/A ptsH (HPr) phosphorylatableprotein (of PTS) Phosphoryl transfer N/A ptsI protein (of PTS)Polypeptide chain (of PTS) N/A Crr

TABLE 7 E.C. Gene Name in Enzyme Function Classification E. coliComments β ketoacyl-acyl carrier protein 2.3.1.179 fabF Decreasefunction, including by synthase I 2.3.1.41 mutation3-OXOACYL-ACP-SYNTHASE II- MONOMER β-ketoacyl-ACP synthase I, 3-2.3.1.41 fabB Decrease function, including by oxoacyl-ACP-synthase I2.3.1.— mutation Malonyl-CoA-ACP transacylase 2.3.1.39 fabD Decreasefunction, including by mutation enoyl acyl carrier protein reductase1.3.1.9, fabI Decrease function, including by 1.3.1.10 mutationβ-ketoacyl-acyl carrier protein 2.3.1.180 fabH Decrease function,including by synthase III mutation Carboxyl transferase subunit α6.4.1.2 accA Increase function subunit Biotin carboxyl carrier protein6.4.1.2 accB Increase function Biotin carboxylase subunit 6.3.4.14 accCIncrease function Carboxyl transferase subunit β 6.4.1.2 accD Increasefunction subunit long chain fatty acyl thioesterase I 3.1.2.2, tesAIncrease function 3.1.1.5 GDP pyrophosphokinase/GTP 2.7.6.5 relADecrease function, including by pyrophosphokinase mutation GDPdiphosphokinase/guanosine- 2.7.6.5, spot Decrease function, including by3′,5′-bis(diphosphate) 3′- 3.1.7.2 mutation diphosphatase

Further with regard to decreasing enzyme function based on Table 7'steachings, any one or a combination of enzyme functions of the followingmay be decreased in a particular embodiment combined with other geneticmodifications described herein: β-ketoacyl-ACP synthase I,3-oxoacyl-ACP-synthase I; Malonyl-CoA-ACP transacylase; enoyl acylcarrier protein reductase; and β-ketoacyl-acyl carrier protein synthaseIII.

Accordingly, as described in various sections above, some compositions,methods and systems of the present invention comprise providing agenetically modified microorganism that comprises both a productionpathway to a selected chemical product, such as 3-HP, and a modifiedpolynucleotide that encodes an enzyme of the fatty acid synthase systemthat exhibits reduced activity, so that utilization of malonyl-CoAshifts toward the production pathway compared with a comparable(control) microorganism lacking such modifications. The methods involveproducing the chemical product using a population of such geneticallymodified microorganism in a vessel, provided with a nutrient media.Other genetic modifications described herein, to other enzymes, such asacetyl-CoA carboxylase and/or NADPH-dependent transhydrogenase, may bepresent in some such embodiments. Providing additional copies ofpolynucleotides that encode polypeptides exhibiting these enzymaticactivities is shown to increase 3-HP production. Other ways to increasethese respective enzymatic activities is known in the art and may beapplied to various embodiments of the present invention. SEQ ID NOs forthese polynucleotides and polypeptides of E. coli are: acetyl-CoAcarboxylase (accABCD, SEQ ID NOs:771-778); and NADPH-dependenttranshydrogenase (SEQ ID NOs:779-782), also referred to as pyridinenucleotide transhydrogenase, pntAB in E. coli).

Also, without being limiting, a first step in some multi-phase methodembodiments of making a chemical product may be exemplified by providinginto a vessel, such as a culture or bioreactor vessel, a nutrient media,such as a minimal media as known to those skilled in the art, and aninoculum of a genetically modified microorganism so as to provide apopulation of such microorganism, such as a bacterium, and moreparticularly a member of the family Enterobacteriaceae, such as E. coli,where the genetically modified microorganism comprises a metabolicpathway that converts malonyl-CoA to 3-HP molecules. For example,genetic modifications may include the provision of at least one nucleicacid sequence that encodes a gene encoding the enzyme malonyl-CoAreductase in one of its bi-functional forms, or that encodes genesencoding a mono-functional malonyl-CoA reductase and an NADH- orNADPH-dependent 3-hydroxypropionate dehydrogenase (e.g., ydfG or mmsBfrom E. coli, or mmsB from Pseudomonas aeruginosa). In either case, whenprovided into an E. coli host cell, these genetic modifications completea metabolic pathway that converts malonyl-CoA to 3-HP. This inoculum iscultured in the vessel so that the cell density increases to a celldensity suitable for reaching a production level of 3-HP that meetsoverall productivity metrics taking into consideration the next step ofthe method. In various alternative embodiments, a population of thesegenetically modified microorganisms may be cultured to a first celldensity in a first, preparatory vessel, and then transferred to thenoted vessel so as to provide the selected cell density. Numerousmulti-vessel culturing strategies are known to those skilled in the art.Any such embodiments provide the selected cell density according to thefirst noted step of the method.

Also without being limiting, a subsequent step may be exemplified by twoapproaches, which also may be practiced in combination in variousembodiments. A first approach provides a genetic modification to thegenetically modified microorganism such that its enoyl-ACP reductaseenzymatic activity may be controlled. As one example, a geneticmodification may be made to substitute for the native enoyl-ACPreductase a temperature-sensitive mutant enoyl-ACP reductase (e.g.,fabI^(Ts) in E. coli). The latter may exhibit reduced enzymatic activityat temperatures above 30 C but normal enzymatic activity at 30 C, sothat elevating the culture temperature to, for example to 34 C, 35 C, 36C, 37 C or even 42 C, reduces enzymatic activity of enoyl-ACP reductase.In such case, more malonyl-CoA is converted to 3-HP or another chemicalproduct than at 30 C, where conversion of malonyl-CoA to fatty acids isnot impeded by a less effective enoyl-ACP reductase.

For the second approach, an inhibitor of enoyl-ACP reductase, or anotherof the fatty acid synthase enzyme, is added to reduce conversion ofmalonyl-CoA to fatty acids. For example, the inhibitor cerulenin isadded at a concentration that inhibits one or more enzymes of the fattyacid synthase system. FIG. 2A depicts relevant pathways and shows threeinhibitors—thiolactomycin, triclosan, and cerulenin, next to the enzymesthat they inhibit. Encircled E coli gene names indicate atemperature-sensitive mutant is available for the polypeptide encoded bythe gene. FIG. 2B provides a more detailed depiction of representativeenzymatic conversions and exemplary E. coli genes of the fatty acidsynthetase system that was more generally depicted in FIG. 2A. Thislisting of inhibitors of microorganism fatty acid synthetase enzymes isnot meant to be limiting. Other inhibitors, some of which are used asantibiotics, are known in the art and include, but are not limited to,diazaborines such as thienodiazaborine, and, isoniazid.

The 3-HP tolerance aspects of the present invention can be used with anymicroorganism that makes 3-HP, whether that organism makes 3-HPnaturally or has been genetically modified by any method to produce3-HP.

As to the 3-HP production increase aspects of the invention, which mayresult in elevated titer of 3HP in industrial bio-production, thegenetic modifications comprise introduction of one or more nucleic acidsequences into a microorganism, wherein the one or more nucleic acidsequences encode for and express one or more production pathway enzymes(or enzymatic activities of enzymes of a production pathway). In variousembodiments these improvements thereby combine to increase theefficiency and efficacy of, and consequently to lower the costs for, theindustrial bio-production production of 3-HP.

Any one or more of a number of 3-HP production pathways may be used in amicroorganism such as in combination with genetic modifications directedto improve 3-HP tolerance. In various embodiments genetic modificationsare made to provide enzymatic activity for implementation of one or moreof such 3-HP production pathways. Several 3-HP production pathways areknown in the art. For example, U.S. Pat. No. 6,852,517 teaches a 3-HPproduction pathway from glycerol as carbon source, and is incorporatedby reference for its teachings of that pathway. This reference teachesproviding a genetic construct which expresses the dhaB gene frontKlebsiella pneumoniae and a gene for an aldehyde dehydrogenase. Theseare stated to be capable of catalyzing the production of 3-HP fromglycerol. However, it is recognized that in some embodiments the carbonsource for a bio-production of 3-HP excludes glycerol as a major portionof the carbon source.

WO 2002/042418 teaches several 3-HP production pathways. This PCTpublication is incorporated by reference for its teachings of suchpathways. Also, FIG. 44 of that publication, which summarizes a 3-HPproduction pathway from glucose to pyruvate to acetyl-CoA to malonyl-CoAto 3-HP, is provided herein. FIG. 55 of that publication, whichsummarizes a 3-HP production pathway from glucose to phosphoenolpyruvate(PEP) to oxaloacetate (directly or via pyruvate) to aspartate toβ-alanine to malonate semialdehyde to 3-HP, is provided herein.Representative enzymes for various conversions are also shown in thesefigures.

FIG. 13, from U.S. Patent Publication No. US2008/0199926, published Aug.21, 2008 and incorporated by reference herein, summarizes theherein-described 3-HP production pathways and other known naturalpathways. More generally as to developing specific metabolic pathways,of which many may be not found in nature. Hatzimanikatis et al. discussthis in “Exploring the diversity of complex metabolic networks,”Bioinformatics 21(8):1603-1609 (2005). This article is incorporated byreference for its teachings of the complexity of metabolic networks.

Further to the 3-HP production pathway summarized in the figures,Strauss and Fuchs (“Enzymes of a novel autotrophic CO₂ fixation pathwayin the phototrophic bacterium Chloroflexus aurantiacus, the3-hydroxyproprionate cycle,” Eur. J. Bichem. 215, 633-643 (1993))identified a natural bacterial pathway that produced 3-HP. At that timethe authors stated the conversion of malonyl-CoA to malonatesemialdehyde was by an NADP-dependant acylating malonate semialdehydedehydrogenase and conversion of malonate semialdehyde to 3-HP wascatalyzed by a 3-hydroxyproprionate dehydrogenase. However, since thattime it has become appreciated that, at least for Chloroflexusaurantiacus, a single enzyme may catalyze both steps (M. Hugler et al.,“Malonyl-Coenzyme A Reductase from Chloroflexus aurantiacus, a KeyEnzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO₂ Fixation,”J. Bacter, 184(9):2404-2410 (2002)).

Accordingly, one production pathway of various embodiments of thepresent invention comprises malonyl-Co-A reductase enzymatic activitythat achieves conversions of malonyl-CoA to malonate semialdehyde to3-HP. As provided in an example herein, introduction into amicroorganism of a nucleic acid sequence encoding a polypeptideproviding this enzyme (or enzymatic activity) is effective to provideincreased 3-HP biosynthesis.

Another 3-HP production pathway is provided in FIG. 14B (FIG. 14Ashowing the natural mixed fermentation pathways) and explained in thisand following paragraphs. This is a 3-HP production pathway that may beused with or independently of other 3-HP production pathways. Onepossible way to establish this biosynthetic pathway in a recombinantmicroorganism, one or more nucleic acid sequences encoding anoxaloacetate alpha-decarboxylase (oad-2) enzyme (or respective orrelated enzyme having such activity) is introduced into a microorganismand expressed. As exemplified in the Examples, which are not meant to belimiting, enzyme evolution techniques are applied to enzymes having adesired catalytic role for a structurally similar substrate, so as toobtain an evolved (e.g., mutated) enzyme (and corresponding nucleic acidsequence(s) encoding it), that exhibits the desired catalytic reactionat a desired rate and specificity a microorganism.

Thus, for various embodiments of the invention the genetic manipulationsto any pathways of the 3HPTGC and any of the 3-HP bio-productionpathways may be described to include various genetic manipulations,including those directed to change regulation of, and therefore ultimateactivity of, an enzyme or enzymatic activity of an enzyme identified inany of the respective pathways. Such genetic modifications may bedirected to transcriptional, translational, and post-translationalmodifications that result in a change of enzyme activity and/orselectivity under selected and/or identified culture conditions. Thus,in various embodiments, to function more efficiently, a microorganismmay comprise one or more gene deletions. For example, as summarized inFIG. 14B for a particular embodiment in E. coli, the genes encodinglactate dehydrogenase (IdhA), phosphate acetyltransferase (pta),pyruvate oxidase (poxB) and pyruvate-formate lyase (pflB) may bedeleted. Such gene deletions are summarized at the bottom of FIG. 14Bfor a particular embodiment, which is not meant to be limiting.Additionally, a further deletion or other modification to reduceenzymatic activity, of multifunctional 2-keto-3-deoxygluconate6-phosphate aldolase and 2-keto-4-hydroxyglutarate aldolase andoxaloacetate decarboxylase (eda in E. coli), may be provided to variousstrains. Further to the latter, in various embodiments combined withsuch reduction of enzymatic activity of multifunctional2-keto-3-deoxygluconate 6-phosphate aldolase and2-keto-4-hydroxyglutarate aldolase and oxaloacetate decarboxylase (edain E. coli), further genetic modifications may be made to increase aglucose transporter (e.g. galP in E. coli) and/or to decrease activityof one or more of heat stable, histidyl phosphorylatable protein (ofPTS) (ptsH (HPr) in E. coli), phosphoryl transfer protein (of PTS) (ptsIin E. coli), and the polypeptide chain of PTS (Crr in E. coli).

Gene deletions may be accomplished by mutational gene deletionapproaches, and/or starting with a mutant strain having reduced or noexpression of one or more of these enzymes, and/or other methods knownto those skilled in the art.

Aspects of the invention also regard skin of multiple geneticmodifications to improve microorganism overall effectiveness inconverting a selected carbon source into a chemical product such as3-HP. Particular combinations are shown, such as in the Examples, toincrease specific productivity, volumetric productivity, titer and yieldsubstantially over more basic combinations of genetic modifications.

Further to FIG. 9 genetic modifications, appropriate additional geneticmodifications can provide further improved production metrics. Forexample, a genetically modified strain is depicted in FIG. 8. Thisstrain comprises genetic modifications for 3-HP production (such asdescribed above in Section VII above), 3-HP tolerance (such as describedbelow), and additional genetic modifications as disclosed herein(including a particular genetic modification regarding the fatty acidsynthase system, not to be limiting, such modifications more generallydisclosed elsewhere herein including in Section VI). In this figureenzyme functions are indicated by indicated enzymatic conversions and/orrepresentative E. coli gene identifiers that encode proteins having suchenzyme functions (except that mer indicates non-E. coli malonyl CoAreductase), deletions are shown by the standard “Δ” before therespective gene identifier, and increased enzymatic activities are shownby underlining (noting that additional targets for modifications are asindicated in the embedded table of the figure). Genes in parentheses arepossible substitutes for or supplements of an enzyme encoded by anothergene also shown along the respective pathway step. Also, the use offabI^(Ts) represents a substitution for the nativenon-temperature-sensitive gene. This is not meant to be limiting; asdescribed elsewhere there are a number of approaches to control andlimit flux to fatty acyl-ACP.

The embodiment of FIG. 8 depicts a number of genetic modifications incombination, however in various embodiments of the present inventionother combinations of the genetic modifications of these enzymaticfunctions may be provided to achieve a desired level of increased rate,titer and yield as to bio-production of a chemical product.

Additional genetic modifications may be provided in a microorganismstrain of the present invention. Many such modifications may be providedto impart a particular phenotype.

As one example, a deletion, of multifunctional 2-keto-3-deoxygluconate6-phosphate aldolase and 2-keto-4-hydroxyglutarate aldolase andoxaloacetate decarboxylase (eda in E. coli), may be provided to variousstrains.

For example, the ability to utilize sucrose may be provided, and thiswould expand the range of feed stocks that can be utilized to produce3-HP or other chemical products. Common laboratory and industrialstrains of E. coli, such as the strains described herein, are notcapable of utilizing sucrose as the sole carbon source. Since sucrose,and sucrose-containing feed stocks such as molasses, are abundant andoften used as feed stocks for the production by microbial fermentation,adding appropriate genetic modifications to permit uptake and use ofsucrose may be practiced in strains having other features as providedherein. Various sucrose uptake and metabolism systems are known in theart (for example, U.S. Pat. No. 6,960,455), incorporated by referencefor such teachings. These and other approaches may be provided instrains of the present invention. The examples provide at least twoapproaches.

Also, genetic modifications may be provided to add functionality forbreakdown of more complex carbon sources, such as cellulosic biomass orproducts thereof, for uptake, and/or for utilization of such carbonsources. For example, numerous cellulases and cellulase-based cellulosedegradation systems have been studied and characterized (see, forexample, and incorporated by reference herein for such teachings,Beguin, P and Aubert, J-P (1994) FEMS Microbial. Rev. 13: 25-58; Ohimi,K. et al. (1997) Biotechnol. Genet. Eng. Rev. 14; 365414).

In addition to the above-described genetic modifications, in variousembodiments genetic modifications also are provided to increase the pooland availability of the cofactor NADPH, and/or, consequently, theNADPH/NADP⁺ ratio. For example, in various embodiments for E. coli, thismay be done by increasing activity, such as by genetic modification, ofone or more of the following genes—pgi (in a mutated form), pntAB,overexpressed, gapA:gapN substitution/replacement, and disrupting ormodifying a soluble transhydrogenase such as sthA, and/or geneticmodifications of one or more of zwf, gnd, and edd.

Any such genetic modifications may be provided to species not havingsuch functionality, or having a less than desired level of suchfunctionality.

More generally, and depending on the particular metabolic pathways of amicroorganism selected for genetic modification, any subgroup of geneticmodifications may be made to decrease cellular production offermentation product(s) selected from the group consisting of acetate,acetoin, acetone, acrylic, malate, fatty acid ethyl esters, isoprenoids,glycerol, ethylene glycol, ethylene, propylene, butylene, isobutylene,ethyl acetate, vinyl acetate, other acetates, 1,4-butanediol,2,3-butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate,2-OH-isobutryate, 3-OH-butyrate, ethanol, isopropanol, D-lactate,L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate,caprolactam, adipic acid, propanol, isopropanol, fusel alcohols, and1,2-propanediol, 1,3-propanediol, formate, fumaric acid, propionic acid,succinic acid, valeric acid, and maleic acid. Gene deletions may be madeas disclosed generally herein, and other approaches may also be used toachieve a desired decreased cellular production of selected fermentationproducts.

X. Separation and Purification of the Chemical Product 3-HP

When 3-HP is the chemical product, the 3-HP may be separated andpurified by the approaches described in the following paragraphs, takinginto account that many methods of separation and purification are knownin the art and the following disclosure is not meant to be limiting.Osmotic shock, sonication, homogenization, and/or a repeated freeze-thawcycle followed by filtration and/or centrifugation, among other methods,such as pH adjustment and heat treatment, may be used to produce a cellfree extract from intact cells. Any one or more of these methods alsomay be employed to release 3-HP from cells as an extraction step.

Further as to general processing of a bio-production broth comprising3-HP, various methods may be practiced to remove biomass and/or separate3-HP from the culture broth and its components. Methods to separateand/or concentrate the 3-HP include centrifugation, filtration,extraction, chemical conversion such as esterification, distillation(which may result in chemical conversion, such as dehydration to acrylicacid, under some reactive-distillation conditions), crystallization,chromatography, and ion-exchange, in various forms. Additionally, cellrupture may be conducted as needed to release 3-HP from the cell mass,such as by sonication, homogenization, pH adjustment or heating. 3-HPmay be further separated and/or purified by methods known in the art,including any combination of one or more of centrifugation,liquid-liquid separations, including extractions such as solventextraction, reactive extraction, two-phase aqueous extraction andtwo-phase solvent extraction, membrane separation technologies,distillation, evaporation, ion-exchange chromatography, adsorptionchromatography, reverse phase chromatography and crystallization. Any ofthe above methods may be applied to a portion of a bio-production broth(i.e., a fermentation broth, whether made under aerobic, anaerobic, ormicroaerobic conditions), such as may be removed from a bio-productionevent gradually or periodically, or to the broth at termination of abio-production event. Conversion of 3-HP to downstream products, such asdescribed herein, may proceed after separation and purification, or,such as with distillation, thin-filin evaporation, or wiped-filmevaporation optionally also in part as a separation means.

For various of these approaches, one may apply a counter-currentstrategy, or a sequential or iterative strategy, such as multi-passextractions. For example, a given aqueous solution comprising 3-HP maybe repeatedly extracted with a non-polar phase comprising an amine toachieve multiple reactive extractions.

When a culture event (fermentation event) is at a point of completion,the spent broth may transferred to a separate tank, or remain in theculture vessel, and in either case the temperature may be elevated to atleast 60° C. for a minimum of one hour in order to kill themicroorganisms. (Alternatively, other approaches to killing themicroorganisms may be practiced.) By spent broth is meant the finalliquid volume comprising the initial nutrient media, cells grown fromthe microorganism inoculum (and possibly including some original cellsof the inoculum), 3-HP, and optionally liquid additions made afterproviding the initial nutrient media, such as periodic additions toprovide additional carbon source, etc. It is noted that the spent brothmay comprise organic acids other than 3-HP, such as for example aceticacid and/or lactic acid.

A centrifugation step may then be practiced to filter out the biomasssolids (e.g., microorganism cells). This may be achieved in a continuousor batch centrifuge, and solids removal may be at least about 80%, 85%,90%, or 95% in a single pass, or cumulatively after two or more serialcentrifugations.

An optional step is to polish the centrifuged liquid through a filter,such as microfiltration or ultrafiltration, or may comprise a filterpress or other filter device to which is added a filter aid such asdiatomaceous earth. Alternative or supplemental approaches to this andthe centrifugation may include removal of cells by a flocculent, wherethe cells floc and are allowed to settle, and the liquid is drawn off orotherwise removed. A flocculent may be added to a fermentation brothafter which settling of material is allowed for a time, and thenseparations may be applied, including but not limited to centrifugation.

After such steps, a spent broth comprising 3-HP and substantially freeof solids is obtained for further processing. By “substantially free ofsolids” is meant that greater than 98%, 99%, or 99.5% of the solids havebeen removed.

In various embodiments this spent broth comprises various ions of salts,such as Na, Cl, SO₄, and PO₄. In some embodiments these ions may beremoved by passing this spent broth through ion exchange columns, orotherwise contacting the spent broth with appropriate ion exchangematerial. Here and elsewhere in this document, “contacting” is taken tomean a contacting for the stated purpose by any way known to personsskilled in the art, such as, for example, in a column, under appropriateconditions that are well within the ability of persons of ordinary skillin the relevant art to determine. As but one example, these may comprisesequential contacting with anion and cation exchange materials (in anyorder), or with a mixed anion/cation material. This demineralizationstep should remove most such inorganic ions without removing the 3-HP.This may be achieved, for example, by lowering the pH sufficiently toprotonate 3-HP and similar organic acids so that these acids are notbound to the anion exchange material, whereas anions, such as Cl andSO₄, that remain charged at such pH are removed from the solution bybinding to the resin. Likewise, positively charged ions are removed bycontacting with cation exchange material. Such removal of ions may beassessed by a decrease in conductivity of the solution. Such ionexchange materials may be regenerated by methods known to those skilledin the art.

In some embodiments, the spent broth (such as but not necessarily afterthe previous demineralization step) is subjected to a pH elevation,after which it is passed through an ion exchange column, or otherwisecontacted with an ion exchange resin, that comprises anionic groups,such as amines, to which organic acids, ionic at this pH, associate.Other organics that do not so associate with amines at this pH (whichmay be over 6.5, over 7.5, over 8.5, over 9.5. over 10.5, or higher pH)may be separated from the organic acids at this stage, such as byflushing with an elevated pH rinse. Thereafter elution with a lower pHand/or elevated salt content rinse may remove the organic acids. Elutingwith a gradient of decreasing pH and/or increasing salt content rinsesmay allow more distinct separation of 3-HP from other organic acids,thereafter simplifying further processing.

This latter step of anion exchange resin retention of organic acids maybe practiced before or after the demineralization step. However, thefollowing two approaches are alternatives to the anion-exchange resinstep.

A first alternative approach comprises reactive extraction (a form ofliquid-liquid extraction) as exemplified in this and the followingparagraphs. The spent broth, which may be at a stage before or after thedemineralization step above, is combined with a quantity of a tertiaryamine such as Alamine336® (Cognis Corp., Cincinnati, Ohio USA) at lowpH. Co-solvents for the Alamine336 or other tertiary amine may be addedand include, but are not limited to benzene, carbon tetrachloride,chloroform, cyclohexane, disobutyl ketone, ethanol, #2, fuel oil,isopropanol, kerosene, n-butanol, isobutanol, octanol, and n-decanolthat increase the partition coefficient when combined with the amine.After appropriate mixing a period of time for phase separationtranspires, after which the non-polar phase, which comprises 3-HPassociated with the Alamine336 or other tertiary amine, is separatedfrom the aqueous phase.

When a co-solvent is used that has a lower boiling point than the3-HP/tertiary amine, a distilling step may be used to remove theco-solvent, thereby leaving the 3-HP-tertiary amine complex in thenon-polar phase.

Whether or not there is such a distillation step, a stripping orrecovery step may be used to separate the 3-HP from the tertiary amine.An inorganic salt, such as ammonium sulfate, sodium chloride, or sodiumcarbonate, or a base such as sodium hydroxide or ammonium hydroxide, isadded to the 3-HP/tertiary amine to reverse the amine protonationreaction, and a second phase is provided by addition of an aqueoussolution (which may be the vehicle for provision of the inorganic salt).After suitable mixing, two phases result and this allows for tertiaryamine regeneration and re-use, and provides the 3-HP in an aqueoussolution. Alternatively, hot water may also be used without a salt orbase to recover the 3HP from the amine.

In the above approach the phase separation and extraction of 3-HP to theaqueous phase can serve to concentrate the 3-HP. It is noted thatchromatographic separation of respective organic acids also can serve toconcentrate such acids, such as 3-HP. In similar approaches othersuitable, non-polar amines, which may include primary, secondary andquaternary amines, may be used instead of and/or in combination with atertiary amine.

A second alternative approach is crystallization. For example, the spentbroth (such as free of biomass solids) may be contacted with a strongbase such as ammonium hydroxide, which results in formation of anammonium salt of 3-HP. This may be concentrated, and then ammonium-3-HPcrystals are formed and may be separated, such as by filtration, fromthe aqueous phase. Once collected, ammonium-3-HP crystals may be treatedwith an acid, such as sulfuric acid, so that ammonium sulfate isregenerated, so that 3-HP and ammonium sulfate result.

Also, various aqueous two-phase extraction methods may be utilized toseparate and/or concentrate a desired chemical product from afermentation broth or later-obtained solution. It is known that theaddition of polymers, such as dextran and glycol polymers, such aspolyethylene glycol (PEG) and polypropylene glycol (PPG) to an aqueoussolution may result in formation of two aqueous phases. In such systemsa desired chemical product may segregate to one phase while cells andother chemicals partition to the other phase, thus providing for aseparation without use of organic solvents. This approach has beendemonstrated for some chemical products, but challenges associated withchemical product recovery from a polymer solution and low selectivitiesare recognized (See “Extractive Recovery of Products from FermentationBroths,” Joong Kyun Kim et al., Biotechnol. Bioprocess Eng.,1999(4)1-11, incorporated by reference for all of its teachings ofextractive recovery methods).

Various substitutions and combinations of the above steps and processesmay be made to obtain a relatively purified 3-HP solution. Also, methodsof separation and purification disclosed in U.S. Pat. No. 6,534,679,issued Mar. 18, 2003, and incorporated by reference herein for suchmethods disclosures, may be considered based on a particular processingscheme. Also, in some culture events periodic removal of a portion ofthe liquid volume may be made, and processing of such portion(s) may bemade to recover the 3-HP, including by any combination of the approachesdisclosed above.

As noted, solvent extraction is another alternative. This may use any ofa number of and/or combinations of solvents, including alcohols, esters,ketones, and various organic solvents. Without being limiting, afterphase separation a distillation step or a secondary extraction may beemployed to separate 3-HP from the organic phase.

The following published resources are incorporated by reference hereinfor their respective teachings to indicate the level of skill in theserelevant arts, and as needed to support a disclosure that teaches how tomake and use methods of industrial bio-production of 3-HP, and alsoindustrial systems that may be used to achieve such conversion with anyof the recombinant microorganisms of the present invention (BiochemicalEngineering Fundamentals, 2^(nd) Ed. J. E. Bailey and D. F. Ollis,McGraw Hill, New York, 1986, entire book for purposes indicated andChapter 9, pp, 533-657 in particular for biological reactor design; UnitOperations of Chemical Engineering, 5^(th) Ed., W. L. McCabe et al.,McGraw Hill, New York 1993, entire book for purposes indicated, andparticularly for process and separation technologies analyses;Equilibrium Staged Separations, P. C. Wankat, Prentice Hall, EnglewoodCliffs, N.J. USA, 1988, entire book for separation technologiesteachings).

XI. Conversion of 3-HP to Acrylic Acid and Downstream Products

As discussed herein, various embodiments described herein are related toproduction of a particular chemical product, 3-hydroxypropionic acid(3-HP). This organic acid, 3-HP, may be converted to various otherproducts having industrial uses, such as but not limited to acrylicacid, esters of acrylic acid, and other chemicals obtained from 3-HP,referred to as “downstream products.” Under some approaches the 3-HP maybe converted to acrylic acid, acrylamide, and/or other downstreamchemical products, in some instances the conversion being associatedwith the separation and/or purification steps. Many conversions to suchdownstream products are described herein. The methods of the inventioninclude steps to produce downstream products of 3-HP.

As a C₃ building block, 3-HP offers much potential in a variety ofchemical conversions to commercially important intermediates, industrialend products, and consumer products. For example, 3-HP may be convertedto acrylic acid, acrylates (e.g., acrylic acid salts and esters),1,3-propanediol, malonic acid, ethyl-3-hydroxypropionate, ethyl ethoxypropionate, propiolactone, acrylamide, or acrylonitrile.

For example, methyl acrylate may be made from 3-HP via dehydration andesterification, the latter to add a methyl group (such as usingmethanol); acrylamide may be made from 3-HP via dehydration andamidation reactions; acrylonitrile may be made via a dehydrationreaction and forming a nitrile moiety; propriolactone may be made from3-HP via a ring-forming internal esterification reaction (eliminating awater molecule); ethyl 3-HP may be made from 3-HP via esterificationwith ethanol; malonic acid may be made from 3-HP via an oxidationreaction; and 1,3-propanediol may be made from 3-HP via a reductionreaction. Also, acrylic acid, first converted from 3-HP by dehydration,may be esterified with appropriate compounds to form a number ofcommercially important acrylate based esters, including but not limitedto methyl acrylate, ethyl acrylate, methyl acrylate, 2-ethylhexylacrylate, butyl acrylate, and lauryl acrylate. Alternatively, 3HP may beesterified to form an ester of 3-HP and then dehydrated to form theacrylate ester.

Additionally, 3-HP may be oligomerized or polymerized to formpoly(3-hydroxypropionate) homopolymers, or co-polymerized with one ormore other monomers to form various co-polymers. Because 3-HP has only asingle stereoisomer, polymerization of 3-HP is not complicated by thestereo-specificity of monomers during chain growth. This is in contrastto (S)-2-Hydroxypropanoic acid (also known as lactic acid), which hastwo (D, L), stereoisomers that must he considered during itspolymerizations.

As will be further described, 3-HP can be converted into derivativesstarting (i) substantially as the protonated form of 3-hydroxypropionicacid; (ii) substantially as the deprotonated form, 3-hydroxypropionate;or (iii) as mixtures of the protonated and deprotonated forms.Generally, the fraction of 3-HP present as the acid versus the salt willdepend on the pH, the presence of other ionic species in solution,temperature (which changes the equilibrium constant relating the acidand salt forms), and to some extent pressure. Many chemical conversionsmay be carried out from either of the 3-HP forms, and overall processeconomics will typically dictate the form of 3-HP for downstreamconversion.

Also, as an example of a conversion during separation, 3-HP in an aminesalt form, such as in the extraction step herein disclosed using Alamine336 as the amine, may be converted to acrylic acid by contacting asolution comprising the 3-HP amine salt with a dehydration catalyst,such as aluminum oxide, at an elevated temperature, such as 170 to 180C, or 180 to 190 C, or 190 to 200 C, and passing the collected vaporphase over a low temperature condenser. Operating conditions, including3-HP concentration, organic amine, co-solvent (if any), temperature,flow rates, dehydration catalyst, and condenser temperature, areevaluated and improved for commercial purposes. Conversion of 3-HP toacrylic acid is expected to exceed at least 80 percent, or at least 90percent, in a single conversion event. The amine may be re-used,optionally after clean-up. Other dehydration catalysts, as providedherein, may be evaluated. It is noted that U.S. Pat. No.7,186,856discloses data regarding this conversion approach, albeit as part of anextractive salt-splitting conversion that differs from the teachingsherein. However, U.S. Pat. No. 7,186,856 is incorporated by referencefor its methods, including extractive salt-splitting, the latter tofurther indicate the various ways 3-HP may be extracted from a microbialfermentation broth.

Further as to embodiments in which the chemical product beingsynthesized by the microorganism host cell is 3-HP, made as providedherein and optionally purified to a selected purity prior to conversion,the methods of the present invention can also be used to produce“downstream” compounds derived from 3-HP, such as polymerized-3-HP(poly-3-HP), acrylic acid, polyacrylic acid (polymerized acrylic acid,in various forms), methyl acrylate, acrylamide, acrylonitrile,propiolactone, ethyl 3-HP, malonic acid, and 1,3-propanediol. Numerousapproaches may be employed for such downstream conversions, generallyfalling into enzymatic, catalytic (chemical conversion process using acatalyst), thermal, and combinations thereof (including some wherein adesired pressure is applied to accelerate a reaction).

As noted, an important industrial chemical product that may be producedfrom 3-HP is acrylic acid. Chemically, one of the carbon-carbon singlebonds in 3-HP must undergo a dehydration reaction, converting to acarbon-carbon double bond and rejecting a water molecule. Dehydration of3-HP in principle can be carried out in the liquid phase or in the gasphase. In some embodiments, the dehydration takes place in the presenceof a suitable homogeneous or heterogeneous catalyst. Suitabledehydration catalysts are both acid and alkaline catalysts. Followingdehydration, an acrylic acid containing phase is obtained and can bepurified where appropriate by further purification steps, such as bydistillation methods, extraction methods, or crystallization methods, orcombinations thereof.

Making acrylic acid from 3-HP via a dehydration reaction may be achievedby a number of commercial methodologies including via a distillationprocess, which may be part of the separation regime and which mayinclude an acid and/or a metal ion as catalyst. More broadly,incorporated herein for its teachings of conversion of 3-HP, and otherβ-hydroxy carbonyl compounds, to acrylic acid and other relateddownstream compounds, is U.S. Patent Publication No. 2007/0219390 A1,published Sep. 20, 2007, now abandoned. This publication lists numerouscatalysts and provides examples of conversions, which are specificallyincorporated herein. Also among the various specific methods todehydrate 3-HP to produce acrylic acid is an older method, described inU.S. Pat. No. 2,469,701 (Redmon). This reference teaches a method forthe preparation of acrylic acid by heating 3-HP to a temperature between130 and 190° C., in the presence of a dehydration catalyst, such assulfuric acid or phosphoric acid, under reduced pressure. U.S. PatentPublication No. 2005/0222458 A1 (Craciun et al.) also provides a processfor the preparation of acrylic acid by heating 3-HP or its derivatives.Vapor-phase dehydration of 3-HP occurs in the presence of dehydrationcatalysts, such as packed beds of silica, alumina, or titania. Thesepatent publications are incorporated by reference for their methodsrelating to converting 3-HP to acrylic acid.

The dehydration catalyst may comprise one or more metal oxides, such asAl₂O₃, SiO₂ or TiO₂. In some embodiments, the dehydration catalyst is ahigh surface area Al₂O₃ or a high surface area silica wherein the silicais substantially SiO₂. High surface area for the purposes of theinvention means a surface area of at least about 50, 75, 100 m²/g, ormore. In some embodiments, the dehydration catalyst may comprise analuminosilicate, such as a zeolite.

For example, including as exemplified from such incorporated references,3-HP may be dehydrated to acrylic acid via various specific methods,each often involving one or more dehydration catalysts. One catalyst ofparticular apparent value is titanium, such as in the form of titaniumoxide, TiO(2). A titanium dioxide catalyst may be provided in adehydration system that distills an aqueous solution comprising 3-HP,wherein the 3-HP dehydrates, such as upon volatilization, converting toacrylic acid, and the acrylic acid is collected by condensation from thevapor phase.

As but one specific method, an aqueous solution of 3-HP is passedthrough a reactor column packed with a titanium oxide catalystmaintained at a temperature between 170 and 190 C and at ambientatmospheric pressure. Vapors leaving the reactor column are passed overa low temperature condenser, where acrylic acid is collected. The lowtemperature condenser may be cooled to 30 C or less, 2 C or less, or atany suitable temperature for efficient condensation based on the flowrate and design of the system. Also, the reactor column temperatures maybe lower, for instance when operating at a pressure lower than ambientatmospheric pressure. It is noted that Example 1 of U.S. PatentPublication No. 2007/0219390, published Sep. 20, 2007, now abandoned,provides specific parameters that employs the approach of this method.As noted, this publication is incorporated by reference for thisteaching and also for its listing of catalysts that may be used in a3-HP to acrylic acid dehydration reaction.

Further as to dehydration catalysts, the following table summarizes anumber of catalysts (including chemical classes) that may be used in adehydration reaction from 3-HP (or its esters) to acrylic acid (oracrylate esters). Such catalysts, some of which may be used in any ofsolid, liquid or gaseous forms, may be used individually or in anycombination. This listing of catalysts is not intended to be limiting,and many specific catalysts not listed maybe used for specificdehydration reactions. Further without being limiting, catalystselection may depend on the solution pH and/or the form of 3-HP in aparticular conversion, so that an acidic catalyst may be used when 3-HPis in acidic form, and a basic catalyst may be used when the ammoniumsalt of 3-HP is being converted to acrylic acid. Also, some catalystsmay be in the form of ion exchange resins.

TABLE 8 Dehydration Catalysts Catalyst by Chemical Class Non-limitingExamples Acids (including H₂SO₄, HCl, titanic acids, metal oxidehydrates, metal sulfates (MSO₄,. where weak and strong) M = Zn, Sn, Ca,Ba, Ni, Co, or other transition metals), metal oxide sulfates, metalphosphates (e.g., M₃, (PO₄)₂, where M = Ca, Ba), metal phosphates, metaloxide phosphates, carbon (e.g., transition metals on a carbon support),mineral acids, carboxylic acids, salts thereof, acidic resins, acidiczeolites, clays, SiO₂/H₃PO₄, fluorinated Al₂O₃, Nb₂O₃/PO₅ ⁻³, Nb₂O₃/SO₄⁻², Nb₂O₅H₂O, phosphotungstic acids, phosphomolybdic acids,silicomolybdic acids, silicotungstic acids, carbon dioxide Bases(including NaOH, ammonia, polyvinylpyridine, metal hydroxides, Zr(OH)₄,and substituted weak and strong) amines Oxides (generally TiO₂, ZrO₂,Al₂O₃, SiO₂, ZnO₂, SnO₂, WO₃, MnO₂, Fe₂O₃, V₂O₅ metal oxides)

As to another specific method using one of these catalysts, concentratedsulfuric acid and an aqueous solution comprising 3-HP are separatelyflowed into a reactor maintained at 150 to 165° C. at a reduced pressureof 100 mm Hg. Flowing, from the reactor is a solution comprising acrylicacid. A specific embodiment of this method, disclosed in Example 1 ofUS2009/0076297, incorporated by reference herein, indicates a yield ofacrylic acid exceeding 95 percent.

Based on the wide range of possible catalysts and knowledge in the artof dehydration reactions of this type, numerous other specificdehydration methods may be evaluated and implemented for commercialproduction.

The dehydration of 3-HP may also take place in the absence of adehydration catalyst. For example, the reaction may be run in the vaporphase in the presence of a nominally inert packing such as glass,ceramic, a resin, porcelain, plastic, metallic or brick dust packing andstill form acrylic acid in reasonable yields and purity. The catalystparticles can be sized and configured such that the chemistry is, insome embodiments, mass-transfer-limited or kinetically limited. Thecatalyst can take the form of powder, pellets, granules, beads,extrudates, and so on. When a catalyst support is optionally employed,the support may assume any physical form such as pellets, spheres,monolithic channels, etc. The supports may be co-precipitated withactive metal species; or the support may be treated with the catalyticmetal species and then used as is or formed into the aforementionedshapes; or the support may be formed into the aforementioned shapes andthen treated with the catalytic species.

A reactor for dehydration of 3-HP may be engineered and operated in awide variety of ways. The reactor operation can be continuous,semi-continuous, or batch. It is perceived that an operation that issubstantially continuous and at steady state is advantageous fromoperations and economics perspectives. The flow pattern can besubstantially plug flow, substantially well-mixed, or a flow patternbetween these extremes. A “reactor” can actually he a series or networkof several reactors in various arrangements.

For example, without being limiting, acrylic acid may be made from 3-HPvia a dehydration reaction, which may be achieved by a number ofcommercial methodologies including via a distillation process, which maybe part of the separation regime and which may include an acid and/or ametal ion as catalyst. More broadly, incorporated herein for itsteachings of conversion of 3-HP, and other β-hydroxy carbonyl compounds,to acrylic acid and other related downstream compounds, is U.S. PatentPublication No. 2007/0219390 A1, published Sep. 20, 2007, now abandoned.This publication lists numerous catalysts and provides examples ofconversions, which are specifically incorporated herein.

For example, including as exemplified from such incorporated references,3-HP may be dehydrated to acrylic acid via various specific methods,each often involving one or more dehydration catalysts. One catalyst ofparticular apparent value is titanium, such as in the form of titaniumoxide, TiO₂. A titanium dioxide catalyst may be provided in adehydration system that distills an aqueous solution comprising 3-HP,wherein the 3-HP dehydrates, such as upon volatilization, converting toacrylic acid, and the acrylic acid is collected by condensation frontthe vapor phase.

As but one specific method, an aqueous solution of 3-HP is passedthrough a reactor column packed with a titanium oxide catalystmaintained at a temperature between 170 and 190° C. and at ambientatmospheric pressure. Vapors leaving the reactor column are passed overa low temperature condenser, where acrylic acid is collected. The lowtemperature condenser may be cooled to 30° C. or less, 20° C. or less,2° C. or less, or at any suitable temperature for efficient condensationbased on the flow rate and design of the system. Also, the reactorcolumn temperatures may be lower, for instance when operating at apressure lower than ambient atmospheric pressure. It is noted thatExample 1 of U.S. Patent Publication No. 2007/0219390, published Sep.20, 2007, now abandoned, provides specific parameters that employs theapproach of this method. As noted, this publication is incorporated byreference for this teaching and also for its listing of catalysts thatmay be used in a 3-HP to acrylic acid dehydration reaction.

Crystallization of the acrylic acid obtained by dehydration of 3-HP maybe used as one of the final separation/purification steps. Variousapproaches to crystallization are known in the art, includingcrystallization of esters.

As noted above, in some embodiments, a salt of 3-HP is converted toacrylic acid or an ester or salt thereof. For example, U.S. Pat. No.7,186,856 (Meng et al.) teaches a process for producing acrylic acidfrom the ammonium salt of 3-HP, which involves a first step of heatingthe ammonium salt of 3-HP in the presence of an organic amine or solventthat is immiscible with water, to form a two-phase solution and splitthe 3-HP salt into its respective ionic constituents under conditionswhich transfer 3-HP from the aqueous phase to the organic phase of thesolution, leaving ammonia and ammonium cations in the aqueous phase. Theorganic phase is then back-extracted to separate the 3-HP, followed by asecond step of heating the 3-HP-containing solution in the presence of adehydration catalyst to produce acrylic acid. U.S. Pat. No. 7,186,856 isincorporated by reference for its methods for producing acrylic acidfrom salts of 3-HP. Various alternatives to the particular approachdisclosed in this patent may be developed for suitable extraction andconversion processes.

Methyl acrylate may be made from 3-HP via dehydration andesterification, the latter to add a methyl group (such as usingmethanol), acrylamide may be made from 3-HP via dehydration andamidation reactions, acrylonitrile may be made via a dehydrationreaction and forming a nitrile moiety, propriolactone may be made from3-HP via a ring-forming internal esterfication reaction (eliminating awater molecule), ethyl-3-HP may be made from 3-HP via esterificationwith ethanol, malonic acid may be made from 3-HP via an oxidationreaction, and 1,3-propanediol may be made from 3-HP via a reductionreaction.

Malonic acid may be produced from oxidation of 3-HP as produced herein.U.S. Pat. No. 5,817,870 (Haas et al.) discloses catalytic oxidation of3-HP by a precious metal selected from Ru, Rh, Pd, Os, Ir or Pt. Thesecan be pure metal catalysts or supported catalysts. The catalyticoxidation can be carried out using a suspension catalyst in a suspensionreactor or using a fixed-bed catalyst in a fixed-bed reactor. If thecatalyst, preferably a supported catalyst, is disposed in a fixed-bedreactor, the latter can be operated in a trickle-bed procedure as wellas also in a liquid-phase procedure. In the trickle-bed procedure theaqueous phase comprising the 3-HP starting material, as well as theoxidation products of the same and means for the adjustment of pH, andoxygen or an oxygen-containing gas can be conducted in parallel flow orcounter-flow. In the liquid-phase procedure the liquid phase and the gasphase are conveniently conducted in parallel flow.

In order to achieve a sufficiently short reaction time, the conversionis carried out at a pH equal or greater than 6, preferably at least 7,and in particular between 7.5 and 9. According to a preferredembodiment, during the oxidation reaction the pH is kept constant,preferably at a pH in the range between 7.5 and 9, by adding a base,such as an alkaline or alkaline earth hydroxide solution. The oxidationis usefully carried out at a temperature of at least 10° C. andmaximally 70° C. The flow of oxygen is not limited. In the suspensionmethod it is important that the liquid and the gaseous phase are broughtinto contact by stirring vigorously. Malonic acid can be obtained innearly quantitative yields. U.S. Pat. No. 5,817,870 is incorporated byreference herein for its methods to oxidize 3-HP to malonic acid.

1,3-Propanediol may be produced from hydrogenation 3-HP as producedherein. U.S. Patent Publication No. 2005/0283029 (Meng et al.) isincorporated by reference herein for its methods to hydrogenation of3-HP, or esters of the acid or mixtures, in the presence of a specificcatalyst, in a liquid phase, to prepare 1,3-propanediol. Possiblecatalysts include ruthenium metal, or compounds of ruthenium, supportedor unsupported, alone or in combination with at least one or moreadditional metal(s) selected from molybdenum, tungsten, titanium,zirconium, niobium, vanadium or chromium. The ruthenium metal orcompound thereof, and/or the additional metal(s), or compound thereof,may be utilized in supported or unsupported form. If utilized insupported form, the method of preparing the supported catalyst is notcritical and can be any technique such as impregnation of the support ordeposition on the support. Any suitable support may be utilized.Supports that may be used include, but are not limited to, alumina,titania, silica, zirconia, carbons, carbon blacks, graphites, silicates,zeolites, aluminosilicate zeolites, aluminosilicate clays, and the like.

The hydrogenation process may be carried out in liquid phase. The liquidphase includes water, organic solvents that are not hydrogenatable, suchas any aliphatic or aromatic hydrocarbon, alcohols, ethers, toluene,decalin, dioxane, diglyme, n-heptane, hexane, xylene, benzene,tetrahydrofuran, cyclohexane, methylcyclohexane, and the like, andmixtures of water and organic solvent(s). The hydrogenation process maybe carried out batch wise, semi-continuously, or continuously. Thehydrogenation process may be carried out in any suitable apparatus.Exemplary of such apparatus are stirred tank reactors, trickle-bedreactors, high pressure hydrogenation reactors, and the like.

The hydrogenation process is generally carried out at a temperatureranging from about 20 to about 250° C., more particularly from about 100to about 200° C. Further, the hydrogenation process is generally carriedout in a pressure range of from about 20 psi to about 4000 psi. Thehydrogen containing gas utilized in the hydrogenation process is,optionally, commercially pure hydrogen. The hydrogen containing gas isusable if nitrogen, gaseous hydrocarbons, or oxides of carbon, andsimilar materials, are present in the hydrogen containing gas. Forexample, hydrogen from synthesis gas (hydrogen and carbon monoxide) maybe employed, such synthesis gas potentially further including carbondioxide, water, and various impurities.

As is known in the art, it is also possible to convert 3-HP to1,3-propanediol using biological methods. For example, 1,3-propanediolcan be created from either 3-HP-CoA or 3-HP via the use of polypeptideshaving enzymatic activity. These polypeptides can be used either invitro or in vivo. When converting 3-HP-CoA to 1,3-propanediol,polypeptides having oxidoreductase activity or reductase activity (e.g.,enzymes from the 1.,1.1.-class of enzymes) can be used. Alternatively,when creating 1,3-propanediol from 3-HP, a combination of a polypeptidehaving aldyhyde dehydrogenase activity (e.g., an enzyme from the1.1.1.34 class) and a polypeptide having alcohol dehydrogenase activity(e.g., an enzyme from the 1.1.1.32 class) can be used.

Another downstream production of 3-HP, acrylonitrile, may be convertedfrom acrylic acid by various organic syntheses, including, by notlimited to the Sohio acrylonitrile process, a single-step method ofproduction known in the chemical manufacturing industry.

Also, addition reactions may yield acrylic acid or acrylate derivativeshaving alkyl or aryl groups at the carbonyl hydroxyl group. Suchadditions may be catalyzed chemically, such as by hydrogen, hydrogenhalides, hydrogen cyanide, or Michael additions under alkalineconditions optionally in the presence of basic catalysts. Alcohols,phenols, hydrogen sulfide, and thiols are known to add under basicconditions. Aromatic amines or amides, and aromatic hydrocarbons, may beadded under acidic conditions. These and other reactions are describedin Ulmann's Encyclopedia of Industrial Chemistry, Acrylic Acid andDerivatives, WileyVCH Verlag GmbH, Wienham (2005), incorporated byreference for its teachings of conversion reactions for acrylic acid andits derivatives.

Acrylic acid obtained front 3-HP made by the present invention may befurther converted to various chemicals, including polymers, which arealso considered downstream products in some embodiments. Acrylic acidesters may be formed from acrylic acid (or directly from 3-HP) such asby condensation esterification reactions with an alcohol, releasingwater. This chemistry described in Monomeric Acrylic Esters, E. H.Riddle, Reinhold, N.Y. (1954), incorporated by reference for itsesterification teachings. Among esters that are formed are methylacrylate, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate,hydroxyethyl acrylate, isobutyl acrylate, and 2-ethylhexyl acrylate, andthese and/or other acrylic acid and/or other acrylate esters may becombined, including with other compounds, to form various known acrylicacid-based polymers. Although acrylamide is produced in chemicalsyntheses by hydration of acrylonitrile, herein a conversion may convertacrylic acid to acrylamide by amidation.

Acrylic acid obtained from 3-HP made by the present invention may befurther converted to various chemicals, including polymers, which arealso considered downstream products in some embodiments. Acrylic acidesters may be formed from acrylic acid (or directly from 3-HP) such asby condensation esterification reactions with an alcohol, releasingwater. This chemistry is described in Monomeric Acrylic Esters, E. H.Riddle, Reinhold, N.Y. (1954), incorporated by reference for itsesterification teachings. Among esters that are formed are methylacrylate, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate,hydroxyethyl acrylate, isobutyl acrylate, and 2-ethylhexyl acrylate, andthese and/or other acrylic acid and/or other acrylate esters may becombined, including with other compounds, to form various known acrylicacid-based polymers. Although acrylamide is produced in chemicalsyntheses by hydration of acrylonitrile, herein a conversion may convertacrylic acid to acrylamide by amidation.

Direct esterification of acrylic acid can take place by esterificationmethods known to the person skilled in the art, by contacting theacrylic acid obtained from 3-HP dehydration with one or more alcohols,such as methanol, ethanol, 1-propanol, 2-propanol, n-butanol,tert-butanol or isobutartol, and heating to a temperature of at least50, 75, 100, 125, or 150° C. The water formed during esterification maybe removed from the reaction mixture, such as by azeotropic distillationthrough the addition of suitable separation aids, or by another means ofseparation. Conversions up to 95%, or more, may be realized, as is knownin the art.

Several suitable esterification catalysts are commercially available,such as from Dow Chemical (Midland, Mich. US). For example, Amberlyst™131Wet Monodisperse gel catalyst confers enhanced hydraulic andreactivity properties and is suitable for fixed bed reactors. Amberlyst™39Wet is a macroreticular catalyst suitable particularly for stirred andslurry loop reactors. Amberlyst™ 46 is a macroporous catalyst producingless ether byproducts than conventional catalyst (as described in U.S.Pat. No. 5,426,199 to Rohm and Haas, which patent is incorporated byreference for its teachings of esterification catalyst compositions andselection considerations).

Acrylic acid, and any of its esters, may be further converted intovarious polymers. Polymerization may proceed by any of heat, light,other radiation of sufficient energy, and free radical generatingcompounds, such as azo compounds or peroxides, to produce a desiredpolymer of acrylic acid or acrylic acid esters. As one example, anaqueous acrylic acid solution's temperature raised to a temperatureknown to start polymerization (in part based on the initial acrylic acidconcentration), and the reaction proceeds, the process frequentlyinvolving heat removal given the high exothermicity of the reaction.Many other methods of polymerization are known in the art. Some aredescribed in Ulmann's Encyclopedia of Industrial Chemistry,Polyacrylamides and Poly(Acrylic Acids), WileyVCH Verlag GmbH, Wienham(2005), incorporated by reference for its teachings of polymerizationreactions.

For example, the free-radical polymerization of acrylic acid takes placeby polymerization methods known to the skilled worker and can be carriedout either in an emulsion or suspension in aqueous solution or anothersolvent. Initiators, such as but not limited to organic peroxides, oftenare added to aid in the polymerization. Among the classes of organicperoxides that may be used as initiators are diacyls,peroxydicarbonates, monoperoxycarbonates, peroxyketals, peroxyesters,dialkyls, and hydroperoxides. Another class of initiators is azoinitiators, which may be used for acrylate polyermization as well asco-polymerization with other monomers. U.S. Pat. Nos. 5,470,928;5,510,307; 6,709,919; and 7,678,869 teach various approaches topolymerization using a number of initiators, including organicperoxides, azo compounds, and other chemical types, and are incorporatedby reference for such teachings as applicable to the polymers describedherein.

Accordingly, it is further possible for co-monomers, such ascrosslinkers, to be present during the polymerization. The free-radicalpolymerization of the acrylic acid obtained from dehydration of 3-HP, asproduced herein, in at least partly neutralized form and in the presenceof crosslinkers is practiced in certain embodiments. This polymerizationmay result in hydrogels which can then be comminuted, ground and, whereappropriate, surface-modified, by known techniques.

An important commercial use of polyacrylic acid is for superabsorbentpolymers. This specification hereby incorporates by reference ModernSuperabsorbent Polymer Technology, Buchholz and Graham (Editors),Wiley-VCH, 1997, in its entirety for its teachings regardingsuperabsorbent polymers components, manufacture, properties and uses.Superabsorbent polymers are primarily used as absorbents for water andaqueous solutions for diapers, adult incontinence products, femininehygiene products, and similar consumer products. In such consumerproducts, superabsorbent materials can replace traditional absorbentmaterials such as cloth, cotton, paper wadding, and cellulose fiber.Superabsorbent polymers absorb, and retain under a slight mechanicalpressure, up to 25 times or their weight in liquid. The swollen gelholds the liquid in a solid, rubbery state and prevents the liquid fromleaking. Superabsorbent polymer particles can be surface-modified toproduce a shell structure with the shell being more highly crosslinked.This technique improves the balance of absorption, absorption underload, and resistance to gel-blocking. It is recognized thatsuperabsorbent polymers have uses in fields other than consumerproducts, including agriculture, horticulture, and medicine.

Superabsorbent polymers are prepared from acrylic acid (such as acrylicacid derived from 3-HP provided herein) and a crosslinker, by solutionor suspension polymerization. Exemplary methods include U.S. Pat. Nos.5,145,906; 5,350,799; 5,342,899; 4,857,610; 4,985,518; 4,708,997;5,180,798; 4,666,983; 4,734,478; and 5,331,059, each incorporated byreference for their teachings relating to superabsorbent polymers.

Among consumer products, a diaper, a feminine hygiene product, and anadult incontinence product are made with superabsorbent polymer thatitself is made substantially from acrylic acid converted from 3-HP madein accordance with the present invention.

Diapers and other personal hygiene products may be produced thatincorporate superabsorbent polymer made from acrylic acid made from 3-HPwhich is bio-produced by the teachings of the present application. Thefollowing provides general guidance for making a diaper thatincorporates such superabsorbent polymer. The superabsorbent polymerfirst is prepared into an absorbent pad that may be vacuum formed, andin which other materials, such as a fibrous material (e.g., wood pulp)are added. The absorbent pad then is assembled with sheet(s) of fabric,generally a nonwoven fabric (e.g., made from one or more of nylon,polyester, polyethylene, and polypropylene plastics) to form diapers.

More particularly, in one non-limiting process, above a conveyer beltmultiple pressurized nozzles spray superabsorbent polymer particles(such as about 400 micron size or larger), fibrous material, and/or acombination of these onto the conveyer belt at designatedspaces/intervals. The conveyor belt is perforated and under vacuum frombelow, so that the sprayed on materials are pulled toward the beltsurface to form a flat pad. In various embodiments, fibrous material isapplied first on the belt, followed by a mixture of fibrous material andthe superabsorbent polymer particles, followed by fibrous material, sothat the superabsorbent polymer is concentrated in the middle of thepad. A leveling roller may be used toward the end of the belt path toyield pads of uniform thickness. Each pad thereafter may be furtherprocessed, such as to cut it to a proper shape for the diaper, or thepad may be in the form of a long roll sufficient for multiple diapers.Thereafter, the pad is sandwiched between a top sheet and a bottom sheetof fabric (one generally being liquid pervious, the other liquidimpervious), such as on a conveyor belt, and these are attached togethersuch as by gluing, heating or ultrasonic welding, and cut intodiaper-sized units (if not previously so cut). Additional features maybe provided, such as elastic components, strips of tape, etc., for fitand ease of wearing by a person.

The ratio of the fibrous material to polymer particles is known toeffect performance characteristics. In some embodiments, this ratio isbetween 75:25 and 90:10 (see U.S. Pat. No. 4,685,915, incorporated byreference for its teachings of diaper manufacture). Other disposableabsorbent articles may be constructed in a similar fashion, such as foradult incontinence, feminine hygiene (sanitary napkins), tampons, etc.(see, for example, U.S. Pat. Nos. 5,009,653, 5,558,656, and 5,827,255incorporated by reference for their teachings of sanitary napkinmanufacture).

Low molecular-weight polyacrylic acid has uses for water treatment,flocculants, and thickeners for various applications including cosmeticsand pharmaceutical preparations. For these applications, the polymer maybe uncrosslinked or tightly crosslinked, depending on the specificapplication. The molecular weights are typically from about 200 to about1,000,000 g/mol. Preparation of these low molecular-weight polyacrylicacid polymers is described in U.S. Pat. Nos. 3,904,685; 4,301,266;2,798,053; and 5,093,472, each of which is incorporated by reference forits teachings relating to methods to produce these polymers.

Acrylic acid may be co-polymerized with one or more other monomersselected from acrylamide, 2-acrylamido-2-methylpropanesulfonic acid,N,N-dimethylacrylamide, N-isopropylacrylamide, methacrylic acid, andmethacrylamide, to name a few. The relative reactivities of the monomersaffect the microstructure and thus the physical properties of thepolymer. Co-monomers may be derived from 3-HP, or otherwise provided, toproduce co-polymers. Ulmann's Encyclopedia of Industrial Chemistry,Polyacrylamides and Poly(Acrylic Acids), WileyVCH Verlag GmbH, Wienham(2005), is incorporated by reference herein for its teachings of polymerand co-polymer processing.

Acrylic acid can in principle be copolymerized with almost anyfree-radically polymerizable monomers including styrene, butadiene,acrylonitrile, acrylic esters, maleic acid, maleic anhydride, vinylchloride, acrylamide, itaconic acid, and so on. End-use applicationstypically dictate the co-polymer composition, which influencesproperties. Acrylic acid also may have a number of optionalsubstitutions on it, and after such substitutions be used as a monomerfor polymerization, or co-polymerization reactions. As a general rule,acrylic acid (or of its co-polymerization monomers) may be substitutedby any substituent that does not interfere with the polymerizationprocess, such as alkyl, alkoxy, aryl, heteroaryl, benzyl, vinyl, allyl,hydroxy, epoxy, amide, ethers, esters, ketones, maleimides,succinimides, sulfoxides, glycidyl and silyl (see U.S. Pat. No.7,678,869, incorporated by reference above, for further discussion). Thefollowing paragraphs provide a few non-limiting examples ofcopolymerization applications.

Paints that comprise polymers and copolymers of acrylic acid and itsesters are in wide use as industrial and consumer products. Aspects ofthe technology for making such paints can be found in U.S. Pat. Nos.3,687,885 and 3,891,591, incorporated by reference for its teachings ofsuch paint manufacture. Generally, acrylic acid and its esters may formhomopolymers or copolymers among themselves or with other monomers, suchas amides, methacrylates, acrylonitrile, vinyl, styrene and butadiene. Adesired mixture of homopolymers and/or copolymers, referred to in thepaint industry as ‘vehicle’ (or ‘binder’) are added to an aqueoussolution and agitated sufficiently to form an aqueous dispersion thatincludes sub-micrometer sized polymer particles. The paint cures bycoalescence of these ‘vehicle’ particles as the water and any othersolvent evaporate. Other additives to the aqueous dispersion may includepigment, filler (e.g., calcium carbonate, aluminum silicate), solvent(e.g., acetone, benzol, alcohols, etc., although these are not found incertain no VOC paints), thickener, and additional additives depending onthe conditions, applications, intended surfaces, etc. In many paints,the weight percent of the vehicle portion may range from about nine toabout 26 percent, but for other paints the weight percent may varybeyond this range.

Acrylic-based polymers are used for many coatings in addition to paints.For example, for paper coating latexes, acrylic acid is used from0.1-5.0%, along with styrene and butadiene, to enhance binding to thepaper and modify rheology, freeze-thaw stability and shear stability. Inthis context, U.S. Pat. Nos. 3,875,101 and 3,872,037 are incorporated byreference for their teachings regarding such latexes. Acrylate-basedpolymers also are used in many inks, particularly UV curable printinginks. For water treatment, acrylamide and/or hydroxy ethyl acrylate arecommonly co-polymerized with acrylic acid to produce lowmolecular-weight linear polymers. In this context, U.S. Pat. Nos.4,431,547 and 4,029,577 are incorporated by reference for theirteachings of such polymers. Co-polymers of acrylic acid with maleic acidor itaconic acid are also produced for water-treatment applications, asdescribed in U.S. Pat. No. 5,135,677, incorporated by reference for thatteaching. Sodium acrylate (the sodium salt of glacial acrylic acid) canbe co-polymerized with acrylamide (which may be derived from acrylicacid via amidation chemistry) to make an anionic co-polymer that is usedas a flocculant in water treatment.

For thickening agents, a variety of co-monomers can be used, such asdescribed in U.S. Pat. Nos. 4,268,641 and 3,915,921, incorporated byreference for description of these co-monomers, U.S. Pat. No. 5,135,677describes a number of co-monomers that can be used with acrylic acid toproduce water-soluble polymers, and is incorporated by reference forsuch description.

Also as noted, some conversions to downstream products may be madeenzymatically. For example, 3-HP may be converted to 3 HP-CoA, whichthen may be converted into polymerized 3-HP with an enzyme havingpolyhydroxyacid synthase activity (EC 2.3.1.-). Also, 1,3-propanediolcan be made using polypeptides having oxidoreductase activity orreductase activity (e.g. , enzymes in the EC 1.1.1.-class of enzymes).Alternatively, when creating 1,3-propanediol from 3-HP, a combination of(1) a polypeptide having aldehyde dehydrogenase activity (e.g., anenzyme from the 1.1.1.34 class) and (2) a polypeptide having alcoholdehydrogenase activity (e.g., an enzyme from the 1.1.1.32 class) can beused. Polypeptides having lipase activity may be used to form esters.Enzymatic reactions such as these may be conducted in vitro, such asusing cell-free extracts, or in vivo.

Thus, various embodiments of the present invention, such as methods ofmaking a chemical, include conversion steps to any such noted downstreamproducts of microbially produced 3-HP, including but not limited tothose chemicals described herein and in the incorporated references (thelatter for jurisdictions allowing this). For example, one embodiment ismaking 3-HP molecules by the teachings herein and further converting the3-HP molecules to polymerized-3-HP (poly-3-HP) or acrylic acid, and suchas from acrylic acid then producing from the 3-HP molecules any one ofpolyacrylic acid (polymerized acrylic acid, in various forms), methylacrylate, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonicacid, 1,3-propanediol, ethyl acrylate, n-butyl acrylate, hydroxypropylacrylate, hydroxyethyl acrylate, isobutyl acrylate, 2-ethylhexylacrylate, and acrylic acid or an acrylic acid ester to which an alkyl oraryl addition is made, and/or to which halogens, aromatic amines oramides, and aromatic hydrocarbons are added.

Also as noted, some conversions to downstream products may be madeenzymatically. For example, 3-HP may be converted to 3-HP-CoA, whichthen may be converted into polymerized 3-HP with an enzyme havingpolyhydroxyacid synthase activity (EC 2.3.1-). Also, 1,3-propanediol canbe made using polypeptides having oxidoreductase activity or reductaseactivity (e.g., enzymes in the EC 1.1.1.-class of enzymes).Alternatively, when creating 1,3-propanediol from 3HP, a combination of(1) a polypeptide having aldehyde dehydrogenase activity (e.g., anenzyme from the 1.1.1.34 class) and (2) a polypeptide having alcoholdehydrogenase activity (e.g., an enzyme from the 1.1.1.32 class) can beused. Polypeptides having lipase activity may be used to form esters.Enzymatic reactions such as these may be conducted in vitro, such asusing cell-free extracts, or in vivo.

Thus, various embodiments of the present invention, such as methods ofmaking a chemical, include conversion steps to any such noted downstreamproducts of microbially produced 3-HP, including but not limited tothose chemicals described herein and in the incorporated references (thelatter for jurisdictions allowing this). For example, one embodiment ismaking 3-HP molecules by the teachings herein and further converting the3-HP molecules to polymerized 3 HP (poly-3-HP) or acrylic acid, and suchas from acrylic acid then producing from the 3-HP molecules any one ofpolyacrylic acid (polymerized acrylic acid, in various forms), methylacrylate, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonicacid, 1,3-propanediol, ethyl acrylate, acrylate, hydroxypropyl acrylate,hydroxyethyl acrylate, isobutyl acrylate, 2-ethylhexyl acrylate, andacrylic acid or an acrylic acid ester to which an alkyl or aryl additionis made, and/or to which halogens, aromatic amines or amides, andaromatic hydrocarbons are added.

Reactions that form downstream compounds such as acrylates oracrylamides can be conducted in conjunction with use of suitablestabilizing agents or inhibiting agents reducing likelihood of polymerformation. See, for example, U.S. Patent Publication No. 2007/0219390A1. Stabilizing agents and/or inhibiting agents include, but are notlimited to, e.g., phenolic compounds (e.g., dimethoxyphenol (DMP) oralkylated phenolic compounds such as di-tert-butyl phenol), quinones(e.g., t-butyl hydroquinone or the monomethyl ether of hydroquinone(MEHQ)), and/or metallic copper or copper salts (e.g., copper sulfate,copper chloride, or copper acetate). Inhibitors and/or stabilizers canbe used individually or in combinations as will be known by those ofskill in the art. Also, in various embodiments, the one or moredownstream compounds is/are recovered at a molar yield of up to about100 percent, or a molar yield in the range from about 70 percent toabout 90 percent, or a molar yield in the range from about 80 percent toabout 100 percent, or a molar yield in the range from about 90 percentto about 100 percent. Such yields may be the result of single pass(batch or continuous) or iterative separation and purification steps ina particular process.

Acrylic acid and other downstream products are useful as commodities inmanufacturing, such as in the manufacture of consumer goods, includingdiapers, textiles, carpets, paint, adhesives, and acrylic glass.

XII. Production of Chemical Products Other Than 3-HP

Disclosures relating to 3-HP are not meant to be limiting, and it isappreciated that other chemical products may be produced frommalonyl-CoA by using the present invention in microorganism host cellsthat comprise production pathways to such chemical products. The variousteaching and combinations of genetic modifications disclosed herein maybe, as appropriate, applied to the microorganisms, methods and systemsthat make 3-HP.

In various embodiments a microorganism cell comprises a metabolicpathway from malonyl-CoA to a selected chemical product, such as 3-HP asparticularly described herein, and means for modulating conversion ofmalonyl-CoA to fatty acyl AC molecules (which thereafter may beconverted to fatty acids) also are provided. Then, when the means formodulating modulate to decrease such conversion, a proportionallygreater number of malonyl-CoA molecules are 1) produced and/or 2)converted via the metabolic pathway from malonyl-CoA to the selectedchemical product.

A metabolic pathway from malonyl-CoA to 3-HP is disclosed herein and isnot meant to be limiting. Other pathways to 3-HP are known in the artand may be utilized to produce 3-HP, including in combination with anycombination of tolerance genetic modifications, as described herein. Asshown in an example herein, addition of such genetic modificationsrelated to the 3HPTGC unexpectedly increase specific; productivity at3-HP levels below toxic levels. Any production pathway that produces3-HP may be combined with genetic modifications of the 3-HPTGC andachieve the specific and/or volumetric productivity metrics disclosedherein.

As to other metabolic pathways for chemical products other than 3-HP,various metabolic pathways for chemical products produced frommalonyl-CoA are known to exist in particular organisms (E.g., see<<www.metacyc.org>>), and genetic recombination techniques may be usedto provide into a selected microorganism cell the polynucleotides thatencode various polynucleotides that catalyze conversions along arespective metabolic pathway. Particular methods of geneticrecombination are disclosed herein, and general references teaching suchmethods also are known to those skilled in the art and also referred toherein, so that one skilled in the art of genetic engineering reasonablymay construct such microorganism cell based on these teachings.Alternatively a wild-type microorganism cell comprising such metabolicpathway may be utilized as a starting cell for use in the presentinvention, such as for genetic modification and/or the methods andsystems disclosed and claimed herein.

XIII. Disclosed Embodiments Are Non-Limiting

While various embodiments of the present invention have been shown anddescribed herein, it is emphasized that such embodiments are provided byway of example only. Numerous variations, changes and substitutions maybe made without departing from the invention herein in its variousembodiments. Specifically, and for whatever reason, for any grouping ofcompounds, nucleic acid sequences, polypeptides including specificproteins including functional enzymes, metabolic pathway enzymes orintermediates, elements, or other compositions, or concentrations statedor otherwise presented herein in a list, table, or other grouping (suchas metabolic pathway enzymes shown in a figure), unless clearly statedotherwise, it is intended that each such grouping provides the basis forand serves to identify various subset embodiments, the subsetembodiments in their broadest scope comprising every subset of suchgrouping by exclusion of one or more members (or subsets) of therespective stated grouping. Moreover, when any range is describedherein, unless clearly stated otherwise, that range includes all valuestherein and all sub-ranges therein.

Also, and more generally, in accordance with disclosures, discussions,examples and embodiments herein, there may be employed conventionalmolecular biology, cellular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. (See, e.g., Sambrook and Russell, “MolecularCloning: A Laboratory Manual,” Third Edition 2001 (volumes 1-3), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal CellCulture, R. I. Freshney, ed., 1986.) These published resources areincorporated by reference herein for their respective teachings ofstandard laboratory methods found therein. Such incorporation, at aminimum, is for the specific teaching and/or other purpose that may benoted when citing the reference herein. If a specific teaching and/orother purpose is not so noted, then the published resource isspecifically incorporated for the teaching(s) indicated by one or moreof the title, abstract, and/or summary of the reference. If no suchspecifically identified teaching, and/or other purpose may be sorelevant, then the published resource is incorporated in order to morefully describe the state of the art to which the present inventionpertains, and/or to provide such teachings as are generally known tothose skilled in the art, as may be applicable. However, it isspecifically stated that a citation of a published resource herein shallnot be construed as an admission that such is prior art to the presentinvention. Also, in the event that one or more of the incorporatedpublished resources differs from or contradicts this application,including but not limited to defined terms, term usage, describedtechniques, or the like, this application controls. Subject matter inthe Examples is incorporated into this section to the extent not alreadypresent.

EXAMPLES

The examples herein provide some examples, not meant to be limiting, ofcombinations of genetic modifications and supplement additions. Thefollowing examples include both actual examples and prophetic examples.

Unless indicated otherwise, temperature is in degrees Celsius andpressure is at or near atmospheric pressure at approximately 5,340 feet(1,628 meters) above sea level. It is noted that work done at externalanalytical and synthetic facilities is not conducted at or nearatmospheric pressure at approximately 5,340 feet (1,628 meters) abovesea level. Examples 11A and 11C were conducted at a contract laboratory,not at the indicated elevation. All reagents, unless otherwiseindicated, are obtained commercially. Species and other phylogenicidentifications are according to the classification known to a personskilled in the art of microbiology.

The names and city addresses of major suppliers are provided herein. Inaddition, as to Qiagen products, the DNeasy® Blood and Tissue Kit, Cat.No. 69506, is used in the methods for genomic DNA preparation; theQIAprep® Spin (“mini prep”), Cat. No. 27106, is used for plasmid DNApurification, and the QIAquick® Gel Extraction Kit, Cat. No. 28706, isused for gel extractions as described herein.

Example 1 Construction of Plasmids Expressing Malonyl-CoA Reductase(mcr)

The nucleotide sequence for the malonyl-CoA reductase gene fromChloroflexus aurantiacus was codon-optimized for E. coli according to aservice from DNA2.0 (Menlo Park, Calif. USA), a commercial DNA genesynthesis provider. This gene sequence (SEQ ID NO:803) incorporated anEcoRI restriction site before the start codon and was followed by aHindIII restriction site. To addition, a ribosomal binding site wasplaced in front of the start codon. This gene construct was synthesizedby DNA2.0 and provided in a pI206 vector backbone (SEQ ID NO:804).Plasmid DNA pI206 containing the synthesized mcr gene was subjected toenzymatic restriction digestion with the enzymes EcoRI and HindIIIobtained from New England BioLabs (Ipswich, Mass. USA) according tomanufacturer's instructions. The digestion mixture was separated byagarose gel electrophoresis and the appropriate DNA fragment recoveredas described in the Common Methods Section. An E. coli cloning strainhearing pKK223-aroH was obtained as a kind gift from the laboratory ofProf. Ryan T. Gill from the University of Colorado at Boulder. Culturesof this strain bearing the plasmid were grown and plasmid DNA preparedas described in the Common Methods Section. Plasmid DNA was digestedwith the restriction endonucleases EcoRI and HindIII obtained from NewEngland Biolabs (Ipswich, Mass. USA) according to manufacturer'sinstructions. This digestion served to separate the aroH reading framefrom the pKK223 backbone. The digestion mixture was separated by agarosegel electrophoresis, and the agarose gel slice containing the DNA piececorresponding to the backbone of the pKK223 plasmid was recovered asdescribed in the Common Methods Section.

Purified DNA fragments corresponding to the mcr gene and pK223 vectorbackbone were ligated and the ligation product was transformed andelectroporated according to manufacturer's instructions. The sequence ofthe resulting vector termed pKK223-mcr was confirmed by routinesequencing performed by a commercial provider (SEQ ID NO:003).pKK223-mcr confers resistance to ampicillin and contains the mcr gene ofC. aurantiacus under control of a P_(tac) promoter inducible in E. colihosts by IPTG.

To express the mcr gene under the regulation of other promoters besidesthe P_(tac) on pKK223, the synthetic mcr gene was transferred to otherplasmids. Plasmid pTrc-P_(tre)-mcr was based on pTrcHisA (Invitrogen,Carlsbad, Calif.; Catalog Number V360-20) and the expression of mcr isdirected by the P_(tre) IPTG-inducible promoter. The inducer-independentP_(ta1A) promoter is based on sequences upstream of the E. coli talAgene. The nucleotide sequence of this promoter, placed immediatelyupstream of the initiator ATG codon of the synthetic mcr gene, is listedas SEQ ID NO:805.

The P_(ta1A):mcr construct was incorporated by PCR into a pSC-B vector(Stratagene Corporation, La Jolla, Calif., USA), which was propagated inan E. coli stock, the plasmid DNA purified according to methodsdescribed elsewhere herein. The P_(ta1A):mcr region inpSC-B-P_(ta1A):mcr was transferred to a plasmid vector, pSMART-HCamp(Lucigen Corporation, Middleton, Wis., catalog number 40041-2, GenBankAF399742) by PCR using vector primers, M13F and M13R. The fragmentgenerated by PCR was cloned into pSMART-HCamp according to themanufacturer's protocol resulting in plasmid pSMART(HC)Amp-P_(ta1A)-mcr(SEQ ID NO:806) in which mcr expression does not require induction withIPTG.

Example 2 Construction of a Plasmid Expressing Transhydrogenase (pntAB)

A fusion of the inducer-independent E. coli promoter derived from thetpiA gene (P_(tpiA)) and the pyridine nucleotide transhydrogenase genes,pntAB, (SEQ ID NO:779 and SEQ ID NO:781) was created by amplifying thetpiA promoter region and pntAB region from genomic E. coli K12 DNA bypolymerase chain reactions. For the pntAB genes, the region wasamplified using the pntAB forward primer GGGAACCATGGCAATTGGCATACCAAG(SEQ ID NO:807, noting that all primers disclosed herein are artificialsequences) containing a NcoI site that incorporates the initiator Metfor the protein sequence of pntA and the pntAB reverse primerGGGTTACAGAGCTTTCAGGATTGCATCC (SEQ ID NO:808). Likewise, the P_(tpiA)region was amplified using the forward primer GGGAACGGCGGGGAAAAACAAACGTT(SEQ ID NO:809) and the reverse primer GGTCATGGTAATTCTCCACGCTTATAAGC(SEQ NO:810) containing a NcoI restriction site. Polymerase chainreaction products were purified using a PCR purification kit from QiagenCorporation (Valencia, Calif., USA) using the manufacturer'sinstructions. Following purification, the products were subjected toenzymatic restriction digestion with the enzyme NcoI. Restrictionenzymes were obtained front New England BioLabs (Ipswich, Mass. USA),and used according to manufacturer's instructions. The digestionmixtures were separated by agarose gel electrophoresis, and visualizedunder UV transillumination as described in the Common Methods Section.Agarose gel slices containing the DNA fragment corresponding to theamplified pntAB gene product and the P_(tpiA) product were excised fromthe gel and the DNA recovered with a gel extraction kit from Qiagen usedaccording to manufacturer's instructions. The recovered products wereligated together with T4 DNA ligase (New England BioLabs, Ipswich, Mass.USA) according to manufacturer's instructions.

Because the ligation reaction can result in several different products,the desired product corresponding to the P_(tpiA) fragment ligated tothe pntAB genes was amplified by polymerase chain reaction and isolatedby a second gel purification. For this polymerase chain reaction, theforward primer was GGGAACGGCGGGGAAAAACAAACGTT (SEQ ID NO:809), and thereverse primer was GGGTTACAGAGCTTTCAGGATTGCATCC (SEQ ID NO:808), and theligation mixture was used as template. The digestion mixtures wereseparated by agarose gel electrophoresis, and visualized under UVtransillumination as described the Common Methods Section. Agarose gelslices containing the DNA piece corresponding to the amplifiedP_(tpiA)-pntAB fusion was cut from the gel and the DNA recovered with astandard gel extraction protocol and components from Qiagen according tomanufacturer's instructions. This extracted DNA was inserted into apSC-B vector using the Blunt PCR Cloning kit obtained from StratageneCorporation (La Jolla, Calif., USA) using the manufacturer'sinstructions. Colonies were screened by colony polymerase chainreactions. Plasmid DNA from colonies showing inserts of correct sizewere cultured and miniprepped using a standard miniprep protocol andcomponents from Qiagen according to the manufacturer's instruction.Isolated plasmids were checked by restriction digests and confirmed bysequencing. The sequenced-verified isolated plasmids produced with thisprocedure were designated pSC-B-P_(tpiS):pntAB.

The P_(tpiA):pntAB region in pSC-B-P_(tpiA):pntAB was transferred to apBT-3 vector (SEQ ID NO:811) which provides a broad host range origin ofreplication and a chloramphenicol selection marker. To achieve thisconstruct, a fragment from pBT-3 vector was produced by polymerase chainamplification using the forward primer AACGAATTCAAGCTTGATATC (SEQ IDNO:812), and the reverse primer GAATTCGTTGACGAATTCTCT (SEQ ID NO:813),using pBT-3 as template. The amplified product was subjected totreatment with DpnI to restrict the methylated template DNA, and themixture was separated by agarose gel electrophoresis, and visualizedunder UV transillumination as described in the Common Methods Section.The agarose gel slice containing the DNA fragment corresponding toamplified pBT-3 vector product was cut from the gel and the DNArecovered with a standard gel extraction protocol and components fromQiagen according to manufacturer's instructions. The P_(tpiA):pntABinsert in pSC-B-P_(tpiA):pntAB was amplified using a polymerase chainreaction with the forward primer GGAAACAGCTATGACCATGATTAC (SEQ IDNO:814) and the reverse primer TTGTAAAACGACGGCCAGTGAGCGCG (SEQ IDNO:815. Both primers were 5′ phosphorylated.

The PCR product was separated by agarose gel electrophoresis, andvisualized under UV transillumination as described in the Common MethodsSection. Agarose gel slices containing the DNA fragment corresponding tothe amplified P_(tpiA):pntAB insert was excised from the gel and the DNArecovered with a standard gel extraction protocol and components fromQiagen according to manufacturer's instructions. This insert DNA wasligated into the pBT-3 vector prepared as described herein with T4 DNAligase obtained from New England Biolabs (Bedford, Mass., USA),following the manufacturer's instructions. Ligation mixtures weretransformed into E. coli 10G cells obtained from Lucigen Corp accordingto the manufacturer's instructions. Colonies were screened by colonypolymerase chain reactions. Plasmid DNA from colonies showing inserts ofcorrect size were cultured and purified using a standard miniprepprotocol and components from Qiagen according to the manufacturer'sinstruction. Isolated plasmids were checked by restriction digests andconfirmed by sequencing. The sequenced-verified isolated plasmidproduced with this procedure was designated pBT-3-P_(tpiA):pntAB (SEQ IDNO:816).

Example 3 Construction of a Plasmid Expressing acetyl-GSA Carboxylase(accABCD)

A plasmid carrying two operons able to express the components theacetyl-CoA carboxyltransferase complex from E. coli was constructed byDNA2.0 (Menlo Park, Calif. USA), a commercial DNA gene synthesisprovider. This construct incorporated the DNA sequences of the accA andaccD genes under control of an inducer-independent promoter derived fromthe E. coli tpiA gene, and the DNA sequences of the accB and accC genesunder control of an inducer-independent promoter derived from the E.coli rpiA genes. Each coding sequence was preceded by a ribosome-bindingsequence. The designed operons were provided in a pJ251 vector backboneand was designated pJ251:26385 (SEQ ID NO:817).

The tpiA promoter of the pJ251:26385 plasmid was altered to providebetter expression. This modification was incorporated by amplifying thepJ251:26385 plasmid with the forward primer GCGGGGCAGGAGGAAAAACATG (SEQID NO:818) and the reverse primerGCTIATAAGCGAATAAAGGAAGATGGCCGCCCCGCAGGGCAG (SEQ ID NO:819). Each ofthese primers were synthesized with a 5′ phosphorylation modification.The resulting PCR product was separated by agarose gel electrophoresis,and the appropriate DNA fragment recovered as described in the CommonMethods Section. The recovered product was self-ligated with T4 DNAligase obtained from New England BioLabs (Ipswich, Mass. USA) anddigested with DpnI according to manufacturer's instructions. Plasmid DNAfrom colonies showing inserts of correct size were cultured and purifiedusing a standard miniprep protocol and components from Qiagen accordingto the manufacturer's instruction. Isolated plasmids were checked byrestrictions digests and confirmed by sequencing. The sequenced-verifiedisolated plasmids produced with this procedure were designatedpJ251(26385)-P_(tpiA):accAD-P_(tpiA):accBC (SEQ ID NO:820).

Example 4 Construction of Plasmids Expressing Genes Related to the 3-HPToleragenic Complex

The examples of plasmid construction for plasmids that comprise genesexpressing polypeptides exhibiting enzymatic activity of the 3HPTGC areincorporated from WO 20101011874, published Jan. 28, 2010. Although manysingle or combination of genetic modifications of the 3HPTGC may beprovided in a particular embodiment so as to increase 3-HP tolerance,only a few are provided in the examples. This is not meant to belimiting.

Example 5 Construction of Specific Strains that Produce3-hydroxypropionic Acid

According to the respective combinations indicated in the followingtable, the plasmids described herein were introduced into the respectivebase strains. All plasmids were introduced at the same time viaelectroporation using standard methods. Transformed cells were gown onthe appropriate media with antibiotic supplementation and colonies wereselected based on their appropriate growth on the selective media. Themcr expression plasmid pKK223-mcr was transformed into E. coli DF40(Hfr, garB10, fhuA22, ompF627, fadL701, relA1, pitA10, spoT1, rruB-2,pgi-2, creC527) or E. coli JP1111 (Hfr, galE45(GalS), LAM-, fabI392(ts,temperature-sensitive), relA1, spoT1, thi-1) as described in the CommonMethods Section. As is known in the art, the strains DF40 and JP1111 aregenerally available E. coli strains, available from sources includingthe Yale Coli Genetic Stock Collection (New Haven, Conn. USA). Strainscarrying multiple compatible plasmids were constructed from these mcrtransformants by preparing cells competent for transformation byelectroporation as described in the Common Methods Section andtransforming with the additional plasmids. Transformants weresubsequently selected for on media containing the appropriatecombination of antibiotics.

TABLE 9 Strain names and characteristics Strain name Host PlasmidsKX3_0001 DF40 pKK223-mcr JX3_0077 JP1111 pKK223-mcr JX3_0087 JP1111pkk223-mcr + pBT-3-PtpiA:pntAB JX3_0097 JP1111 pkk223-mcr +pJ251(26385)PtpiA:accAD-PrpiA:accBC JX3_0098 JP1111 pKK223-mcr +pJ251(26385)PtpiA:accAD-PrpiA:accBC + pBT-3-PtpiA:pntAB

Example 6 Production of 3-hydroxypropionic Acid

3-HP production by KX3_0001 was demonstrated at 100-mL scale infed-hatch (rich) or AM2 (minimal salts) media. Cultures were startedfrom freezer stocks by standard practice (Sambrook and Russell, 2001)into 50 mL of LB media plus 100 μg/mL ampicillin and grown to stationaryphase overnight at 37° C. with rotation at 225 rpm. Five ml of thisculture were transferred to 100 ml of fed-batch or AM2 media plus 40g/L. glucose, 100 μg/ml ampicillin, 1 mM IPTG in triplicate 250-mlbaffled flasks, and incubated at 37° C., 225 rpm To monitor cell growthand 3-HP production by these cultures, samples (2 ml) were withdrawn atdesignated time points for optical density measurements at 600 nm(OD₆₀₀, 1 cm pathlength) and pelleted by centrifugation at 12,000rpm for5 min and the supernatant collected for analysis of 3-HP production asdescribed under “Analysis of cultures for 3-HP production” in the CommonMethods section. Dry cell weight (DCW) is calculated as 0.33 times themeasured OD₆₀₀ value, based on baseline DCW to OD₆₀₀ determinations. Alldata are the average of triplicate cultures. For comparison purposes,the specific productivity is calculated from the averaged data at the24-h time point and expressed as g 3-HP produced per gDCW. Production of3-HP by strain KX3_0001 in fed-batch medium is shown in the followingtable. Under these conditions, the specific productivity after 24 h is0.0041 g 3-HP per gDCW.

TABLE 10 Production of 3-HP by KX3_0001 in fed-batch medium Time (hr)3HP (g/L) OD₆₀₀ 0 0.002 0.118 3 0.002 0.665 4 0.005 1.44 6 0.008 2.75 80.009 3.35 24 0.008 5.87

Example 7 Effect on 3-HP Production of Increased malonyl-CoA PrecursorPools by Inhibition of Fatty Acid Synthesis

As described herein, certain chemicals are known to inhibit variousenzymes of the fatty acid synthase system, some of which are used asantibiotics given the role of fatty acid synthesis in membranemaintenance and growth, and microorganism growth. Among these inhibitorsis cerulenin, which inhibits the KASI β-ketoacyl-ACP synthase (e.g.,fabB in E. coli). To further evaluate approaches to modulate and shiftmalonyl-CoA utilization in microorganisms that comprise productionpathways to a selected chemical product, here 3-HP, wherein malonyl-CoAis a substrate in that pathway, addition of cerulenin during a culturewas evaluated.

Pathways downstream of malonyl-CoA are limited to fatty acidbiosynthesis and 3HP production (when a pathway to the latter viamalonyl-CoA exists or is provided in a cell). This experiment isdesigned to determine how to control the use of malonyl-CoA pools in 3HPproduction strains and further improve the rate of 3HP production. It ishypothesized that by inhibiting fatty acid biosynthesis and regulatingmalonyl-CoA pools, flux through the pathway will be shifted toward 3HPproduction. A diagram of the possible carbon flow through malonyl-CoA incurrent 3HP production pathways is shown in FIG. 9. A representativeinhibitor has been selected that both interrupt fatty acid elongationand disrupt a futile cycle that recaptures the malonate moiety back tothe acetyl-CoA pool.

Production by strain KX3_0001 in batch medium in the presence of 10μg/ml cerulenin is shown in Table 11. In the presence of the inhibitor,internal pools of the malonyl-CoA precursor are proposed to increasethus leading to increased production of 3-HP. As may be seen bycomparison to the results without cerulenin (Table 5), substantiallymore 3-HP is produced at every time point, and the specific productivityat 24 h is 0.128 g 3-HP per gDCW, a 31-fold increase relative to theresults without cerulenin.

TABLE 11 Production of 3-HP by KX3_0001 in fed-batch medium and thepresence of 10 μg/ml cerulenin 3HP (g/L) OD₆₀₀ 0.002 0.118 0.002 0.7240.020 1.59 0.060 2.80 0.090 3.45 0.200 4.73

Example 8 Effect on 3-HP Production of Increased malonyl-CoA PrecursorPools using Temperature-Sensitive Fatty Acid Synthesis Mutants

An alternative approach to increasing internal malonyl-CoA pools is touse genetic mutations rather than chemical inhibitors. Whileinactivating mutations in the genes encoding fatty acid synthesisfunctions are usually lethal and thus not obtainable, conditionalmutants, such as temperature-sensitive mutants, have been described (deMendoza, D., and Cronan, J. E., Jr. (1983) Trends Biochem. Sci., 8,49-52). For example, a temperature-sensitive mutation in the fabI gene,encoding enoyl-ACP reductase, of strain JP1111 (genotype fabI392(ts))has relatively normal activity at reduced temperature, such as 30 C, andbecomes non-permissive, likely through denaturation and inactivation, atelevated temperature, such that when cultured at 37 to 42 C amicroorganism only comprising this temperature sensitive mutant as itsenoyl-ACP reductase will produce substantially less fatty acids andphospholipids. This leads to decreased or no growth. However, it washypothesized that when such mutant is provided in a genetically modifiedmicroorganism that also comprises a production pathway, such as to 3-HP,from malonyl-CoA, effective culture methods involving elevating, culturetemperature can result in increased 3-HP specific productivity.

Production of 3-HP by strain JX3_0077 in fed-batch medium at a constanttemperature of 30° C. and by a culture subjected to a temperature shiftfrom 30° C. to 42° C. is shown in Table 12. The temperature shift isdesigned to inactivate the enoyl-ACP reductase, hence eliminating theaccumulation of fatty acid which in turn increases the internalmalonyl-CoA pool. Substantially more 3-HP is produced at every timepoint, and the specific productivity at 24 h by the temperature-shiftedculture is 1.15 g 3-HP per gDCW, a greater than 100-fold increase overthe specific productivity of 0.011 g 3-HP per gDCW by the culturemaintained constantly at 30° C. This increased productivity of 3-HP bythe culture in which the enoyl-ACP reductase is inactivated by elevatedtemperature supports the view that shifting of malonyl-CoA utilizationleads to increased 3-HP production.

TABLE 12 Production of 3-HP by JX3_0077 in fed-batch medium Constant 30°C. Shifted to 42° C. Time (hr) 3HP (g/L) OD₆₀₀ 3HP (g/L) OD₆₀₀ 0 0 0.0650.0007 0.068 3 0.003 0.273 0.004 0.25 4 0.010 0.409 0.037 0.79 6 0.0301.09 0.096 0.91 8 0.016 1.81 0.193 0.81 24 0.014 3.8 0.331 0.87

Table 13 shows the 3-HP production by strain JX3_0087 which carried aplasmid overexpressing the transhydrogenase gene in addition to aplasmid carrying the mcr gene. In the culture maintained at a constanttemperature of 30° C., a specific productivity of 0.085 g 3-HP per gDCWin 24 h was attained. This is significantly higher than the specificproductivity of JX3_0077 which does not carry the overexpressedtranshydrogenase gene (Table 7). The specific productivity of thetemperature-shifted culture of JX3_0087 was 1.68 g 3-HP per gDCW, a20-fold increase over the specific productivity of the culturemaintained constantly at 30° C. In which the enoyl-ACP reductase was notinactivated.

TABLE 13 Production of 3-HP by JX3_0087 in fed-batch medium Constant 30°C. Shifted to 42° C. Time (hr) 3HP (g/L) OD₆₀₀ 3HP (g/L) OD₆₀₀ 0 0 0.0080 0.004 3 0.0007 0.008 0.0007 0.011 4 0 0.04 0.002 0.063 6 0.0007 0.050.009 0.193 8 0.003 0.157 0.050 0.257 24 0.003 0.107 0.455 0.820

Table 14 shows the 3-HP production by strain JX3_0097 which carried aplasmid overexpressing genes encoding the acetyl-CoA carboxylase complexin addition to a plasmid carrying the mcr gene. In the culturemaintained at a constant temperature of 30° C., a specific productivityof 0.0068 g 3-HP per gDCW in 24 h was attained. This specificproductivity is similar to that attained by strain JX3_0077 in whichacetyl-CoA carboxylase is not overexpressed. The specific productivityof the temperature-shifted culture of JX3_0097 was 0.29 g 3-HP per gDCW,a 42-fold increase over the specific productivity of the culturemaintained constantly at 30° C. In which the enoyl-ACP reductase was notinactivated

TABLE 14 Production of 3-HP by JX3_0097 in fed-batch medium Constant 30°C.* Shifted to 42° C.* Time (hr) 3HP (g/L) OD₆₀₀ 3HP (g/L) OD₆₀₀ 0 0.0160 0.014 4 0.004 0.3 0.004 0.31 5 0.36 0.006 0.59 6 0.65 0.062 1.51 80.006 1.46 0.178 1.91 24 0.006 2.66 0.176 1.87

Fed-batch medium, a rich medium, may contain components that serve asfatty acid precursors and thus may reduce the demand for malonyl-CoA.Thus the production of 3-HP by the strains derived from JP1111 in AM2, aminimal medium was verified. As shown in Table 15, 3-HP was produced byJX3_0077 in AM2 medium. A specific productivity of 0.024 g 3-HP per gDCWin 24 h was obtained by the culture maintained constantly at 30° C.,approximately twice the value obtained in fed-batch medium. Thetemperature shifted culture attained a specific productivity of 1.04 g3-HP per gDCW over 24 h, a 44-fold increase compared to the specificproductivity of the culture maintained constantly at 30° C., againindicating that conditional inactivation of the enoyl-ACP reductaseincreased the internal malonyl-CoA pool and hence increased the 3-HPproduction, as envisioned by the inventors.

TABLE 15 Production of 3-HP by JX3_0077 in AM2 medium Constant 30° C.Shifted to 42° C. Time (hr) 3HP (g/L) OD₆₀₀ 3HP (g/L) OD₆₀₀ 0 0 0.066 00.063 4 0.002 0.360 0.002 0.40 5 0.004 0.253 0.015 0.39 6 0.004 0.4130.1 0.68 8 0.005 0.476 0.2 0.71 24 0.008 1.03 0.25 0.73

Production of 3-HP in AM2 medium by strain JX3_0087, which carried aplasmid overexpressing the transhydrogenase gene in addition to aplasmid carrying the mcr gene, is shown. In the JX3_0087 culturemaintained at a constant temperature of 30° C., a specific productivityof 0.018 g 3-HP per gDCW in 24 h was attained. In contrast to resultsobtained in fed-hatch medium, this value is not higher than the specificproductivity obtained in AM2 with strain JX3_0077 which does not carrythe overexpressed transhydrogenase gene (Table 15). The specificproductivity of the temperature-shifted culture of JX3_0087 was 0.50 g3-HP per gDCW, a 27 fold increase over the specific productivity of theculture maintained constantly at 30° C. In which the enoyl-ACP reductasewas not inactivated.

TABLE 16 Production of 3-HP by JX3_0087 in AM2 Constant 30° C. Shiftedto 42° C. Time (hr) 3HP (g/L) OD₆₀₀ 3HP (g/L) OD₆₀₀ 0 0 0.08 0 0.086 40.002 0.363 0.002 0.380 5 0.002 0.273 0.011 0.360 6 0.003 0.297 0.0500.520 8 0.005 0.467 0.100 0.607 24 0.006 1.0 0.112 0.683

Table 17 shows the 3-HP production in AM2 medium by strain JX3_0097which carried a plasmid overexpressing genes encoding the acetyl-CoAcarboxylase complex in addition to a plasmid carrying the mcr gene. Inthe culture maintained at a constant temperature of 30° C., a specificproductivity of 0.021 g 3-HP per gDCW in 24 h was attained. Thisspecific productivity is similar to that attained by strain JX3_0077 inwhich acetyl-CoA carboxylase is not overexpressed. The specificproductivity of the temperature-shifted culture of JX13_0097 was 0.94 g3-HP per gDCW in 24 h, a 45-fold increase over the specific productivityof the culture maintained constantly at 30° C. In which the enoyl-ACPreductase was not inactivated.

TABLE 17 Production of 3-HP by JX3_0097.0 in AM2 Constant 30° C. Shiftedto 42° C. Time (hr) 3HP (g/L) OD₆₀₀ 3HP (g/L) OD₆₀₀ 0 0 0.085 0.0010.085 4 0.002 0.500 0.003 0.483 5 0.003 0.287 0.015 0.473 6 0.005 0.4170.073 0.510 8 0.005 0.520 0.198 0.590 24 0.013 1.91 0.192 0.620

The effect of combining the plasmids expressing mcr (malonyl-CoAreductase), pntAB (transhydrogenase), and accABCD (acetyl CoAcarboxylase complex) in the same organism was tested by constructingstrain JX3_0098. The Table above shows the production of 3-HP by thisstrain in AM2 medium. A specific productivity of 0.54 g 3-HP per gDCW in24 h was obtained in the culture maintained constantly at 30° C.,representing a >20-fold increase over strains carrying mcr alone or mcrwith either pntAB or accABCD, but not both. Shifting, the temperature toinactivate enoyl-ACP reductase resulted in a specific productivity of2.01 g 3-HP per gDCW in 24 h, a further 3.8-fold increase. Thus thecombination of overexpression of pntAB and of accABCD, plus theinactivation of enoyl-ACP reductase via the temperature-sensitivefabI^(Ts) allele, resulted in an approximately 500-fold increase inspecific productivity of 3-HP by mcr-bearing cells (specificproductivity of 2.01 vs. 0.0041 g 3-HP per gDCW in 24 h).

TABLE 18 Production of 3-HP by JX3_0098.0 in AM2 medium Constant 30° C.Shifted to 42° C. Time (hr) 3HP (g/L) OD₆₀₀ 3HP (g/L) OD₆₀₀ 0 0.0070.117 0 0.13 4 0.013 0.303 0.017 0.47 5 0.017 0.600 0.060 0.75 6 0.0330.730 0.107 0.87 8 0.053 0.9107 0.263 0.81 24 0.670 3.790 0.577 0.81

Example 9 Sequence of the fabI^(Ts) Mutation

The nature of the exact sequence change in the fobI^(Ts) allele carriedby strains JP1111 was reconfirmed. Confirmation of this change allowstargeted mutagenesis to generate alternative strains with differenttemperature sensitivities and mutants with stabilities intermediatebetween wild type and the fabI392 temperature-sensitive allele, allowinggrowth at a constant temperature higher than 30° C. while providing thebenefit of increased internal malonyl-CoA pools. To confirm the DNAsequence of this segment of the chromosome of a wild type (BW25113) andthe JP1111 mutant E. coli, chromosomal DNA was prepared from thesestrains. These DNA were used as templates in a PCR reaction withprimers:

FW043 SEQ ID NO: 821 ATGGGTTTTCTTTCCGG FW047 SEQ ID NO: 822TTATTTCAGTTCGAGTTCG

Thermocyler conditions for the PCR were: 95° C., 10 min; 30 cycles of95° C., 10 s; 47° C. increasing to 58° C., 30 s; 72° C., 1 min; followedby a final incubation at 72° C. for 5 min. The PCR product was separatedon an agarose gel and the appropriate sized fragment recovered asdescribed in the Common Methods Section, and sequenced using primers:

FW044 SEQ ID NO: 823 CTATCCATCGCCTACGGTATC FW045 SEQ ID NO: 824CGTTGCAATGGCAAAAGC FW046 SEQ ID NO: 825 CGGCGGTTTCAGCATTGC

A comparison of the DNA sequence obtained from the fabI392 (SEQ IDNO:769) and wild type strains reveals a single difference between thealleles of C at position 722 of the wild type gene to T (see FIG. 4A),leading to a protein change of Ser at codon 241 to Phe (See FIG. 4B).These changes are identical to those found by Bergler, H., Hogenauer,G., and Turnowsky, F., J. Gen. Microbiol. 138:2093-2100 (1992).

The identification of the affected residue at codon 241 indicates thattargeted mutagenesis at this coclon, for example to amino acid residuessuch as Trp, Tyr, His, Ile, or other amino acids other than Ser or Phe,may result in fabI alleles with different properties than the fabI392originally isolated in JP1111. Targeted mutagenesis at codons near tocodon 241 may also be contemplated to obtain the desired fabI mutantswith altered properties.

Example 10 Effect on 3-HP Production of Overexpression of Genes from the3-HP Toleragenic Complex

A series of strains were constructed carrying plasmids that express mcr(pTrc-P_(tre)-mcr or pSMART(HC)Amp-P_(ta1A)-mcr) alone or withcompatible plasmids carrying representative genes from the 3-HPtoleragenic complex (pJ61-aroG, pJ61-thrA, pACYC177-cynTS, pJ61-cynTS).Table 19 categorizes the strains and their characteristics.

TABLE 19 Strain name and characteristics of strain carrying plasmidsbearing toleragenic complex genes Strain name Host Plasmids JX3_0118JP1111 pTrc-P_(trc)-mcr JX3_0110 JP1111 pTrc-P_(trc)-mcr + pJ61-aroGJX3_0111 JP1111 pTrc-P_(trc)-mcr + pJ61-thrA JX3_0112 JP1111pTrc-P_(trc)-mcr + pACYC177-cynTS JX3_0113 JP1111 pTrc-P_(trc)-mcr +pJ61-cynTS JX3_0104 JP1111 pSMART(HC)Amp-P_(talA)-mcr JX3_0114 JP1111pSMART (HC)Amp-P_(talA)-mcr + pJ61-aroG JX3_0119 JP1111 pSMART(HC)Amp-P_(talA)-mcr + p15A empty vector JX3_0115 JP1111 pSMART(HC)Amp-P_(talA)-mcr + pJ61-thrA JX3_0116 JP1111 pSMART(HC)Amp-P_(talA)-mcr + pACYC177-cynTS JX3_0117 JP1111 pSMART(HC)Amp-P_(talA)-mcr + pJ61-cynTS JX3_0119 JP1111 pSMART(HC)Amp-P_(talA)-mcr + p15A empty vector

Production of 3-HP by strains carrying pTrc-P_(tre)-mcr without and withplasmids carrying genes from the 3-HP toleragenic complex (3HPTGC) isshown in Table 20. 3-HP production was carried out as in Example 6except cultures were maintained at constant 30° C., and strains wereevaluated based on their specific productivity after 24 hr. As shown inTable 20, the specific productivity of strain JX3_0118, which differsfrom strain JX3_0077 only in the nature of the IPTG-inducible plasmid,was 0.19 g 3-HP/gDCW in 24 h compared to 0.011 g 3-HP per gDCW byJX3_0077. This 17-fold increase in specific productivity by the culturemaintained at a constant 30° C. is attributable to increased stabilityand mcr expression by pTrc-P_(1rc)-mcr.

Expression of genes from the 3-HP toleragenic complex further increasesproductivity of 3-HP. Expression of aroG in IX3_0110 resulted in a2.3-fold increase, expression of thrA in JX3_0111 resulted in a 2.2-foldincrease, and expression of cynTS in JX3_0112 resulted in a 10.6-foldincrease in specific productivity in 24 hr.

TABLE 20 Specific Productivity Time 3 HP (g 3-HP/gDCW) Strain (hr) (g/L)OD₆₀₀ at 24 h JX3_0118 4 0.01 0.21 6 0.03 0.50 8 0.06 0.87 24 0.19 3.10.19 JX3_0110 4 0.05 0.28 6 0.09 0.51 8 0.15 0.70 24 0.40 2.8 0.43JX3_0111 4 0.04 0.26 6 0.08 0.51 8 0.13 0.62 24 0.33 2.4 0.42 JX3_0112 40.04 0.26 6 0.10 0.50 8 0.20 0.64 24 0.60 0.90 2.02 JX3_0113 4 0.01 0.066 0.02 0.20 8 0.02 0.24 24 0.08 2.2 0.11

Similar results were obtained in strains carrying mcr expressed bypSMART (HC)Amp-P_(ta1A)-mcr and additional plasmids carrying genes fromthe 3-HP toleragenic complex. 3-HP production was carried out as inExample 6 except cultures were maintained at constant 30° C., andstrains were evaluated based on their specific productivity after 24 hr.Strains carrying the mcr expression plasmid alone (JX3_(———)0104), orwith an empty control vector (JX3_0119) had specific productivities of0.062 or 0.068 g 3-HP per gDCW in 24 hr, respectively. Expression ofaroG in JX3_0114 resulted in a 2.4-fold increase, expression of thrA inJX3_(——)0115 resulted in a 2.6-fold increase, and expression of cynTS inJX3_0116 or JX3_0117 resulted in a 2.1-fold increase in specificproductivity in 24 hr compared to strain JX3_0119. Thus overexpressionof representative genes from the 3-HP toleragenic complex significantlyincreased the specific productivity of 3-HP even at levels of excreted3-HP much below those at which the tolerance effects of these genes werefirst identified. This is an unexpected beneficial result.

TABLE 21 Production of 3-HP by strains carrying pSMART(HC)Amp-P_(talA)-mcr and plasmids bearing genes from the 3-HPtoleragenic complex Specific Productivity Time 3 HP (g 3-HP/gDCW) Strain(hr) (g/L) OD₆₀₀ at 24 h JX3_0104 4 0.01 0.01 6 0.01 0.30 8 0.02 0.80 240.04 1.94 0.062 JX3_0119 4 0.01 0.11 6 0.01 0.4 8 0.02 0.92 24 0.04 1.790.68 JX3_0114 4 0.03 0.19 6 0.04 0.18 8 0.05 0.2 24 0.13 2.38 0.17JX3_0115 4 0.03 0.08 6 0.03 0.25 8 0.04 0.32 24 0.09 1.55 0.18 JX3_01164 0.03 0.13 6 0.04 0.30 8 0.05 0.40 24 0.10 2202 0.15 JX3_0117 4 0.040.18 6 0.05 0.31 8 0.07 0.73 24 0.11 2.4 0.14

Example 11 Effect on Volumetric 3-HP Production in 1 L Fermentations, ofIncreased malonyl-coA Precursor Pools using Temperature Sensitive FattyAcid Synthesis Mutants

Four 1 L fed batch fermentation experiments were carried out using thestrain JX3_0098. Briefly, seed cultures were started and grown overnightin LB media (Luria Broth) and used to inoculate four 1 L New Brunswickfermentation vessels. The first vessel contained defined AM2 medium at30° C., IPTG induction was added at 2 mM at an OD₆₀₀nm of 2, additionalglucose feed was initiated when glucose was depleted to between 1-2 g/L.The temperature was shifted 37° C. over 1 hr at target OD of 10. A highglucose feed rate was maintained at >3 g/L/hr until glucose began toaccumulate at concentrations greater than 1 g/L at which time feed ratewas varied to maintain residual glucose between 1 and 10 g/L. The secondvessel contained defined AM2 medium at 30° C., IPTG induction was addedat 2 mM at an OD₆₀₀nm of 2, additional glucose feed was initiated whenglucose was depleted to 0 g/L. The temperature was shifted 37° C. over 1hr at target OD of 10. The glucose feed rate was maintained less than orequal to 3g/L/hr. The third vessel contained rich medium at 30° C. IPTGinduction was added at 2 mM at an OD₆₀₀nm of 2, additional glucose feedwas initiated when glucose was depleted to 1-2 g/L. The temperature wasshifted 37° C. over 1 hr at target OD of 10. A high glucose feed ratewas maintained at >3 g/L/hr until glucose began to accumulate atconcentrations greater than 1g/L at which time feed rate was varied tomaintain residual glucose between 1 and 10 g/L. The fourth vesselcontained rich medium at 30° C., IPTG induction was added at 2 mM at anOD₆₀₀nm of 2, additional glucose feed was initiated when glucose wasdepleted to 0 g/L. The temperature was shifted 37° C. over 1 hr attarget OD of 10. The glucose feed rate was maintained less than or equalto 3 g/L/hr.

Growth profiles are shown in FIG. 5, arrows indicate the initiation ofthe temperature shift. All fermentation vessels were maintained atpH=7.4 by the controlled addition of 50% v/v ammonium hydroxide (FisherScientific). All vessels were maintained at least 20% dissolved oxygenby aeration with sparged filtered air. Samples were taken for opticaldensity measurements as well as HPLC analysis for 3-HP concentration.(Refer to common methods). Maximum volumetric productivities reached2.99 g/L/hr. In addition, the figures demonstrate the correlationbetween the 3-4 hour average biomass concentration and 3-4 hr averagevolumetric productivity rates in these 4 vessels.

Example 11A Production of 3-HP in 250 Liter Fermentations

Examples of two fed batch fermentations in a 250 liter volume stainlesssteel fermentor were carried out using the strain BX3_0240, the genotypeof which is described elsewhere herein. A two stage seed process wasused to generate inoculum for the 250 L fermentor. In the first stage,one ml of glycerol stock of the strain was inoculated into 100 ml of TBmedium (Terrific Broth) in a shake flask and incubated at 30° C. untilthe OD₆₀₀ was between 3 and 4. In the second stage, 85 ml of the shakeflask culture was aseptically transferred to a 14 L New Brunswickfermentor containing 8 L of TB medium and grown at 30° C. and 500 rpmagitation until the OD₆₀₀ was between 5 and 6. The culture from the 14 Lfermentor was used to aseptically inoculate the 250 L volume bioreactorcontaining defined FM5 medium (see Common Methods Section) at 30° C. sothat the post-inoculation volume was 155 L.

In the first fermentation, induction was effected by adding IPTG to afinal concentration of 2 mM at an OD₆₀₀ of 20. Glucose feed (consistingof a 700 g/L glucose solution) was initiated when the residual glucosein the fermentor was 10-15 g/L. The feed rate was adjusted to maintainthe residual glucose between 10 and 15 g/L until about the last 6 hoursof the fermentation when the feed rate was reduced so that the residualglucose at harvest was <1 g/L to facilitate 3-HP recovery. Three hoursafter induction, the temperature was shifted to 37° C. over 1 hour. Atthe time the temperature shift was initiated, the dissolved oxygen (DO)set point was changed from 20% of air saturation to a point where the DOwas maintained between 2-4% of air saturation. The fermentation brothwas harvested 48 hours after inoculation. The final broth volume was169.5 liters.

The second fermentation was run identically to the first examplefermentation described above except for the following differences:induction with IPTG was effected at an OD₆₀₀ of 15, the residual glucose(after the glucose feed was started) ranged between 3-30 g/L, and thefermentation broth was harvested at 38.5 hours after inoculation so thatthe final residual glucose concentration was 25 g/L. The final brothvolume was 167 liters.

Each fermentation broth was maintained at a pH of approximately 7.4 bythe controlled addition of anhydrous ammonia gas. Dissolved oxygen wasmaintained at the desired levels by aeration with sparged,sterile-filtered air. Samples were taken for optical densitymeasurements as well as HPLC analysis for 3-HP concentration. In thefirst fermentation, the maximum biomass concentration was 12.0 g drycell weight/L and the biomass concentration at harvest was 11.4 g drycell weight/L. The maximum 3-HP titer in this fermentation was 20.7 g/L.In the second fermentation, the maximum biomass concentration was 10.2 gdry cell weight/L and the biomass concentration at harvest was 9.5 g drycell weight/L. The maximum 3-HP titer in this fermentation was 20.7 g/L.

Example 11B Effect of Growth Medium on 3-HP Production in 1 LFermentations

Eight 1 L fed batch fermentation experiments were carried out using thestrain B X3_0240. Seed culture was started from 1 ml of glycerol stockof the strain inoculated into 400 ml of TB medium (Terrific Broth) in ashake flask and incubated at 30° C. until the OD₆₀₀ was between 5 and 6.The shake flask culture was used to aseptically inoculate each 1 Lvolume bioreactor so that the post-inoculation volume was 653 ml in eachvessel.

Fermentors 1 and 2 contained defined FM3 medium. Fermentors 3-5contained defined FM4 medium. Fermentors 6-8 contained defined FM5medium. All media formulations are listed in the Common Methods Section.In each fermentor, the initial temperature was 30° C.

Induction was effected by adding IPTG to a final concentration of 2 mMat OD₆₀₀ values of 15-16. Glucose feed (consisting of a 500 g/L glucosesolution for FM3 and FM5 media and 500 g/L glucose plus 75 mM MgSO₄ forFM4) was initiated when the residual glucose in the fermentor was about10 g/L. The feed rate was adjusted to maintain the residual glucose >3g/L (the exception was fermentor 8 in which the residual glucosetemporarily reached 0.1 g/L before the feed rate was increased). Threehours after induction, the temperature was shifted to 37° C. over 1hour. At the time the temperature shift was initiated, the dissolvedoxygen (DO) set point was changed from 20% of air saturation to 1% ofair saturation. The fermentations were stopped 48 hours afterinoculation.

The broth of each fermentor was maintained at a pH of approximately 7.4by the controlled addition of a pH titrant. The pH titrant for FM3medium was 5 M. NaOH and for FM4 and FM5 it was a 50:50 mixture ofconcentrated ammonium hydroxide and water. Dissolved oxygen wasmaintained at the desired levels by sparging with sterile-filtered air.Samples were taken for optical density measurements as well as HPLCanalysis for 3-HP concentration. The maximum biomass concentration andthe biomass concentration at harvest as well as the maximum 3-HP titerin each fermentor are summarized in the Table 22 below.

TABLE 22 Maximum Biomass Conc. Maximum Fermentor Growth Biomass Conc. atHarvest 3HP Titer No. Medium (g DCW/L) (g DCW/L) (g/L) 1 FM3 8.7 8.712.3 2 FM3 9.6 9.5 16.7 3 FM4 10.9 10.9 20.7 4 FM4 11.5 11.5 18.3 5 FM411.3 11.3 22.1 6 FM5 11.3 11.3 35.2 7 FM5 11.2 11.0 34.0 8 FM5 11.6 10.631.2

Example 11C Effect of Batch Phosphate Concentration on 3-HP Productionin 1 L Fermentations

Four 1 L fed batch fermentation experiments were carried out using thestrain BX3_0240. Seed culture was started from 1 ml of glycerol stock ofthe strain inoculated into 400 ml of TB medium (Terrific Broth) in ashake flask and incubated at 30° C. until the OD₆₀₀ was between 5 and 7.The shake flask culture was used to aseptically inoculate each 1 Lvolume bioreactor so that the post-inoculation volume was 653 ml in eachvessel.

All fermentors contained defined FM5 growth medium, but each haddifferent initial concentrations of monobasic and dibasic potassiumphosphate. The phosphate concentrations in the batch medium in eachfermentor are summarized in the Table 23. The FM5 media formulation islisted in the Common Methods Section.

TABLE 23 Fermentor K₂HPO₄ conc. in KH₂PO₄ conc. in No. batch medium(g/L) batch medium (g/L) 1 6.1 1.92 2 2.63 1.38 3 0.87 0.14 4 0.0430.070

In each fermentor, the initial temperature was 30° C. Induction waseffected by adding IPTG to a final concentration of 2 mM when the OD₆₀₀values were at the following values: fermentor 1, 15.3; fermentor 2,16.0; fermentor 3, 18.1; fermentor 4, 18.4. Glucose feed (consisting ofa 500 g/L glucose solution for FM3 and FM5 media and 500 g/L, glucoseplus 75 mM MgSO₄ for FM4) was initiated when the residual glucose in thefermentor was about 10 g/L. The feed rate was adjusted to maintain theresidual glucose >6.5 g/L. Three hours after induction, the temperaturewas shifted to 37° C. over 1 hour. At the time the temperature shift wasinitiated, the dissolved oxygen (DO) set point was changed from 20% ofair saturation to 1% of air saturation. The fermentations were stopped48 hours after inoculation.

The broth of each fermentor was maintained at a pH of 7.4 by thecontrolled addition of a 50:50 mixture of concentrated ammoniumhydroxide and water. Dissolved oxygen was maintained at the desiredlevels by sparging with sterile-filtered air. Samples were taken foroptical density measurements as well as HPLC analysis for 3-HPconcentration. The maximum biomass concentration and the biomassconcentration at harvest as well as vie maximum 3-HP titer in eachfermentor are summarized in the Table 24 below.

TABLE 24 Maximum Biomass Conc. Maximum Fermentor Biomass Conc. atHarvest 3HP Titer No. (g DCW/L) (g DCW/L) (g/L) 1 9.6 8.4 23.7 2 11.311.3 27.8 3 14.8 12.9 39.8 4 12.3 10.9 44.1

Example 11D 3-HP Production in 1 L Fermentations

Two 1 L fed batch fermentation experiments were carried out using thestrain BX3_0240. Seed culture was started from 1 mL of glycerol stock ofthe strain inoculated into 100 mL of TB medium (Terrific Broth) in ashake flask and incubated at 30° C. until the OD₆₀₀ was between 5 and 6.The shake flask culture was used to aseptically inoculate (5%volume/volume) each 1 L volume bioreactor so that the post-inoculationvolume was 800 mL in each vessel. The fermentors used in this experimentwere Das Gip fed batch pro parallel fermentation system (DASGIP AG,Julich, Germany, model SR0700ODLS). The fermentation system includedreal-time monitoring and control of dissolved oxygen (% DO), pH,temperature, agitation, and feeding. Fermentors 1 and 2 containeddefined FM5 medium, made as shown in the Common Methods Section exceptthat Citric Acid was added at 2.0 g/L and MgSO0 ₄ was added at 0.40 g/L.In each fermentor, the initial temperature was 30° C. Induction waseffected by adding IPTG to a final concentration of 2 mM at OD₆₀₀ valuesof 17-19, which corresponded to a time past-inoculation of 14.5 hr.Glucose feed (consisting of a 500 g/L glucose solution) was initiatedwhen the residual glucose in the fermentor was about 1 g/L. The feedrate was adjusted to maintain the residual glucose >3 g/L. Three hoursafter induction, the temperature was shifted to 37° C. over 1 hour. Atthe time the temperature shift was initiated, the OTR was set to 40mmol/L-hr by setting airflow and agitation to 1.08 vvm and 1000 rpmrespectively. Compressed air at 2 bar was used as the air feed. Thebroth of each fermenter was maintained at a pH of approximately 7.4 bythe controlled addition of a pH titrant. Two hours subsequent to IPTGinduction, kite pH titrant was changed from 50% NH₄(OH) to 7.4 M NaOH.Samples were taken for optical density measurements as wed as HPLCanalysis for 3-HP concentration. The maximum biomass concentration andthe biomass concentration at harvest as well as the maximum 3-HP titerin each fermenter are summarized in the Table 25 below.

TABLE 25 Yield of 3-HP At Maximum Biomass Conc. Total 3-HP 69 hrsFermentor Biomass Conc. at Harvest (g) at (g3-HP/g No. (g DCW/L) (gDCW/L) 69 hrs glucose) 1 10.5 8.7 49.0 0.46 2 10.5 8.7 47.8 0.46

The following Table 26 provides a summary of concentrations of metabolicproducts obtained in the fermentation broth at the indicated time inhours.

TABLE 26 Time 3-HP Pyruvate Succinate Lactate Replicate (hrs) (g/L)(g/L) (g/L) (g/L) 1 0 0 0.341 0.328 0 1 45 35.128 5.596 0 0 1 69 36.059.179 0 0 2 0 0 0.346 0.376 0 2 45 31.188 8.407 0 0 2 69 35.139 13.143 00 Fumarate Glutamate Glutamine Glycerol Alanine (g/L) (g/L) (g/L) (g/L)(g/L) 0.002 0.006 0 0.563 0.139 0.013 0.959 0 0.160 0.104 0.003 1.77 00.244 0.075 0.002 0.893 0.075 0.471 0.109 0.004 0.796 0 0.347 0.0840.011 1.23 0 0.481 0.077

Example 11E 3-HP Production in 1 L Fermentations

Four 1 L fed batch fermentation experiments were carried out using thestrain BX3_0240. Seed culture was started from 1 ml of glycerol stock ofthe strain inoculated into 100 mL of TB medium (Terrific Broth) in ashake flask and incubated at 30° C. until the OD₆₀₀ was between 5 and 6.The shake flask culture was used to aseptically inoculate (5%volume/volume) each 1 L volume bioreactor so that the post-inoculationvolume was 800 ml in each vessel. The fermentors used in this experimentwere Das Gip fed-batch pro parallel fermentation system (DASGIP AG,Julich, Germany, model SR0700ODLS). The fermentation system includedreal-time monitoring and control of dissolved oxygen (% DO), pH,temperature, agitation, and feeding. All fermentors contained definedFM5 medium, made as shown in the Common Methods Section except thatCitric Acid was added at 2.0 g/L and MgSO₄ was added at 0.40 g/L. Ineach fermentor, the initial temperature was 30° C. Induction waseffected by adding IPTG to a final concentration of 2 mM at OD₆₀₀ valuesof 15-19, which corresponded to a time post-inoculation of 15.75 hr.Glucose feed (consisting of a 500 g/L glucose solution) was initiatedwhen the residual glucose in the fermentor was about 3 g/L. The feedrate was adjusted to maintain the residual glucose >3 g/L. Three hoursafter induction, the temperature was shifted to 37° C. over 1 hour. Thebroth of each fermentor was maintained at a pH of approximately 7.4 bythe controlled addition of a pH titrant 50% NH4(OH). At the time thetemperature shift was initiated, the OTR was changed for each fermentorby varying the agitation and airflow according to Table 27. Compressedair at (2 bar was used as the air feed) Samples were taken for opticaldensity measurements as well as HPLC analysis for 3-HP concentration.The maximum biomass concentration and the biomass concentration atharvest as well as the maximum 3-HP titer in each fermentor aresummarized in the Table 27 below.

TABLE 27 Agitation during Biomass Conc. 3HP Titer Fermentor AirflowProduction at Harvest (g/L) at No. (vvm) (rpm) (g DCW/L) 37 hrs 1 1.081000 8.6 14.9 2 1.08 800 9.0 7.9 3 1.08 600 8.2 0.5 4 1.08 400 5.9 0.5

Example 11F 3-HP Production in 1.8 L Fermentation

A 1.8 L fed batch fermentation experiment was carried out using thestrain BX3_0240. Seed culture was started from 1 ml of glycerol stock ofthe strain inoculated into 105 ml of TB medium (Terrific Broth) in ashake flask and incubated at 30° C. until the OD600 was between 5 and 7.90 ml of the shake flask culture was used to aseptically inoculate 1.71L of FM5 growth medium, except that the phosphate concentrations were0.33 g/L K2HPO4 and 0.17 g/L KH2PO4 in batch medium. The otheringredients in the FM5 media formulation are as listed in the CommonMethods Section. The initial temperature in the fermentor was 30° C.Induction was effected by adding IPTG to a final concentration of 2 mMwhen the OD600 value was at 15.46. Glucose feed (consisting of a 500 g/Lglucose solution) was initiated when the residual glucose in thefermentor was about 10 g/L. The feed rate was adjusted to maintain theresidual glucose >6.5 g/L. Three hours after induction, the temperaturewas shifted to 37° C. over 1 hour. At the time the temperature shift wasinitiated, the dissolved oxygen (DO) set point was changed from 20% ofair saturation to 1% of air saturation. The broth of each fermentor wasmaintained at a pH of 7.4 by the controlled addition of a 50:50 mixtureof concentrated ammonium hydroxide and water. Dissolved oxygen wasmaintained at the desired levels by sparging with sterile-filtered air.Samples were taken for optical density measurements as well as HPLCanalysis for 3-HP concentration. The maximum final biomass concentrationwas 9.84 g/L, the maximum 3-HP titer was 48.4 g/L with a final yieldfrom glucose of 0.53 g 3-HP/g glucose.

Example 12 Strain Construction for Further Evaluations of 3-HPProduction

According to the respective combinations indicated in Table 28 below,the plasmids described herein (e.g., see Example 1) were introduced intothe respective strains. All plasmids were introduced at the same timevia electroporation using standard methods. Transformed cells were grownon the appropriate media with antibiotic supplementation and colonieswere selected based on their appropriate growth on the selective media.As summarized in Table 28, the mcr expression plasmids pTrc-ptrc-mcr orpACYC(kan)-ptalA-mcr were transformed into two strains derived from E.coli BW25113 (F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), lamba-, rph-1,Δ(rhaD-rhaB)568, hsdR514), these strains comprising additionalchromosomal modifications introduced using Gene Bridges technology asdescribed in the Common Methods Section. Strain BX_0590 comprisesadditional deletions of the ldhΔ, pflB, mgsA, and poxB genes. StrainBX_0591 comprises the additional deletions of Strain BX_0590 and anadditional deletion of the ack_pta genes. Transformants weresubsequently selected for on media containing the appropriatecombination of antibiotics.

TABLE 28 Strain name Host Plasmids BX3_0194 BX_0590 PTrc-ptrc-mcrBX3_0195 BX_0591 PTrc-ptrc-mcr BX3_0206 BX_0590 pACYC(kan)-ptalA-mcr

Example 12A Construction of Additional Strains for Evaluation

Part 1: Gene Deletions

The homologous recombination method using Red/ET recombination, asdescribed elsewhere herein, was employed for gene deletion in E. colistrains. This method is known to those of ordinary skill in the art anddescribed in U.S. Pat. Nos. 6,355,412 and 6,509,156, issued to Stewartet al. and incorporated by reference herein for its teachings of thismethod. Material and kits for such method are available from GeneBridges (Gene Bridges GmbH, Heidelberg (formerly Dresden), Germany,<<www.genebridges.com>>), and the method proceeded by following themanufacturer's instructions. The method replaces the target gene by aselectable marker via homologous recombination perforated by therecombinase from λ-phage. The host organism expressing λ-red recombinaseis transformed with a linear DNA product coding for a selectable markerflanked by the terminal regions (generally ˜50 bp, and alternatively upto about ˜300 bp) homologous with the target gene or promoter sequence.The marker is thereafter removed by another recombination step performedby a plasmid vector carrying the FLP-recombinase, or anotherrecombinase, such as Cre.

Specific deletions were constructed by amplification using PCR from theKeio strains carrying particular deletions using primers as specifiedbelow. The Keio collection was obtained from Open Biosystems(Huntsville, Ala. USA 35806). Individual clones may be purchased fromthe Yale Genetic Stock Center (New Haven, Conn. USA 06520). These stainseach contain a kanamycin marker in place of the deleted gene. In caseswhere the desired deletion was not in a Keio strain, for exampleackA-pta, the deletion was constructed by the above-noted recombinationmethod using the kanamycin resistance marker to replace the deletedsequence, followed by selection of a kanamycin resistance clone havingthe deletion. The PCR products were introduced into targeted strainsusing the above-noted recombination method. Combinations of deletionswere generated sequentially to obtain strains as described in thefollowing parts of this example.

TABLE 29 Plasmid Keio Clone Gene Forward Primer Reverse Primer templateNumber Deletion SEQ ID NO: SEQ ID NO: JW1375 ldhA JW0886 pflB 829 842JW5129 mgsA 830 843 JW0855 poxB 831 844 JW2880 serA 832 845 JW4364 arcA833 846 JW4356 trpR 834 847 JW3561 aldB 835 848 JW1412 aldA 836 849JW1293 puuC 837 850 JW2755 relA 838 851 pKD4 spoT 839 852 pKD4 ackA-pta840 853 JW1228 adhE 841 854

Table 31 shows strains having genotypes that comprise deletionsaccording to the methods of this Part.

Part 2: Construction of strains BW_595 and BW_651 having a fabI Mutation

The fabI^(Ts) mutation (Ser241→Phe) in E. coli strain JP1111significantly increases the malonyl-CoA concentration when cells aregrown at the nonpermissive temperature (37° C.) and thus produces more3-HP at this temperature. However, JP1111 is not an ideal strain fortransitioning into pilot and commercial scale, since it is the productof NTG mutagenesis and thus may harbor unknown mutations, carriesmutations in the stringency regulatory factors relA and spoT, and hasenhanced conjugation propensity due to the presence of an Hfr factor.Thus the fabI^(Ts) mutation was moved into strain BX_591, a straindeveloped from the well-characterized BW23115 carrying the additionalmutations ΔldhA, ΔpflB, ΔmgsA, ΔpoxB, Δpta-ack. These mutations weregenerated by the sequential application of the gene deletion methoddescribed in Part 1 above.

The fabI^(Ts) gene with 600 bp of upstream and downstream DNA sequencewas isolated from JP1111 genomic DNA by PCR using primers:

FW056: SEQ ID NO: 855 5′-CCAGTGGGGAGCTACATTCTC; and FW057:SEQ ID NO: 856 5′-CGTCATTCAGATGCTGGCGCGATC.

The FRT::kan::FRT cassette was then inserted at a SmaI site downstreamof the fabI^(Ts) to generate plasmidpSMART(HC)amp_fabI^(Ts)_FRT::kan::FRT. This plasmid was used as templateDNA and the region between primers:

FW043: SEQ ID NO: 857 5′-ATGGGTTTTCTTTCCGG and FW057 (SEQ ID NO: 856)was amplified in a PCR using KOD HS DNA polymerase (Novagen). Thereaction was treated with DpnI to fragment the plasmid template and theamplification fragment was gel-purified and recovered using the DNAClean and Concentrator kit (Zymo Research, Orange, Calif.). StrainBX_591 was transformed with pSLM5 (Datta, S., et al, Gene 379:109-115,2006) and expression of the lambda red genes carried on this plasmidwere induced by incubation at 42″C for 15 min.

Electrocompetent cells were made by standard methods. These cells weretransformed with the amplification fragment bearing thefabI^(Ts)_FRT::kan::FRT cassette and transformant colonies isolated onLB plates containing 35 μg/ml kanamycin at 30° C. Individual colonieswere purified by restreaking, and tested for temperature sensitivity bygrowth in liquid medium at 30° C. and 42° C. Compared to wildtypeparental strain, the strain bearing the faBI^(Ts) allele grows poorly at42° C. but exhibited comparable growth at 30° C. Correct insertion ofthe FRT::kan::FRT marker was verified by colony PCR, and the fabI^(Ts)kan^(R) strain was designated BX_594.

To allow use of the kan^(R) marker on plasmids, the marker incorporatedin the chromosome adjacent to fabI^(Ts) was replaced with a DNA fragmentencoding resistance to zeocin. The zeoR gene was amplified by PCR fromplasmid pJ402 (DNA 2.0, Menlo Park, Calif.) using primers:

HL018: SEQ ID NO: 858 5′-CAGGTTTGCGGCGTCCAGCGGTTATGTAACTACTACTATTCGGCGCGACTTACGCCGCTCCCCGCTCGCGATAATGTGGTAGC; and HL019: SEQ ID NO: 8595′-AATAAAACCAATGATTTGGCTAATGATCACACAGTCCCAGGCAGTAAGACCGACGTCATTCTATCATGCCATACCGCGAA.

The reaction was treated with DpnI and gel-purified as above. StrainBX_594 was transformed with pKD46 (Datsenko and Wanner, Proc, Natl.Acad. Sci. USA 96: 6640-6645, 2000) and the lambda red genes carried onthis plasmid were induced by the addition of L-arabinose to 1 mM for 2hr. Electrocompetent cells were made by standard methods (e.g., Sambrookand Russell, 2001). These cells were transformed with the zeoR fragmentand transformants selected for on LB plates formulated without NaCl andwith 25 μg/ml zeocin. Plates were kept in the dark by wrapping inaluminum foil, and incubated at 30° C. A zeocin-resistant,kanamycin-sensitive strain isolated by this method was designatedBX_595. Retention of the fabI^(Ts) allele was confirmed by growth asabove.

Strain BX_651 was constructed by transferring the fabI^(Ts)-zeoRcassette from BX_595 to strain BW25113 which does not carry mutations inmetabolic genes. A DNA fragment carrying this cassette was obtained byPCR using BX_595 chromosomal DNA and primers FW043 (see above) and SEQID NO:860 FW65: 5′-GAGATAAGCCTGAAATGTCGC.

The PCR product was purified and concentrated using the DNA Clean andConcentrator kit (Zymo Research, Orange, Calif.). Strain BW25113 wastransformed with pRedD/ET (Gene Bridges GMBH, Heidelberg, Germany) andthe lambda red genes carried on this plasmid were induced by theaddition of L-arabinose to 5 mM for 2 hr. Electrocompetent cells weremade by standard methods, and transformed with the fabI^(Ts)-zeoR DNAfragment. Transformants were plated as above on zeocin, and clonesbearing the temperature-sensitive allele verified by growth at 30° C.and 42° C. as described above.

Part 3: Promoter Replacement for Selected Genes in Chromosome

The homologous recombination method described elsewhere herein wasemployed to replace promoters of various genes. As noted, use of Red/ETrecombination is known to those of ordinary skill in the art anddescribed in U.S. Pat. Nos. 6,355,412 and 6,509,156, issued to Stewartet al. and incorporated by reference herein for its teachings of thismethod. Material and kits for such method are available from GeneBridges (Gene Bridges GmbH, Heidelberg, Germany,<<www.genebridges.com>>), and the method may proceed by following themanufacturer's instructions. The method involves replacement of thetarget gene (or, in this case, a promoter region) by a selectable markervia homologous recombination performed by the recombinase from λ-phage.The host organism expressing λ-red recombinase is transformed with alinear DNA product coding for a selectable marker flanked by theterminal regions (generally ˜50 bp, and alternatively up to about ˜300bp) homologous with the target gene or promoter sequence. The marker canthen be removed by another recombination step performed by a plasmidvector carrying the FLP-recombinase, or another recombinase, such asCre. This method was used according to manufacturer's instructions.Template sequences, each comprising end sequences to achieve therecombination to replace a native promoter for the indicated gene ofinterest, the desired replacement promoter, and an antibiotic markersequence, were synthesized by an outside manufacturer (Integrated DNATechnologies, Coralville, Iowa). These sequences are designed to replacethe native promoter in front of these genes with a T5 promoter. TheT5-aceEF cassette (SEQ ID NO:863) also includes a zeocin resistancecassette flanked by loxP sites, The T5-pntAB (SEQ ID NO:864), T5-udhA(SEQ ID NO:865) and T5-cynTS (SEQ ID NO:866) cassettes each include ablasticidin resistance cassette flanked by loxP sites. Also, T5-cynTS(SEQ ID NO:866) comprises modified loxP sites in accordance with Lambertet al., AEM 73(4) p 1126-1135.

Each cassette first is used as a template for PCR amplification togenerate a PCR product using the primers CAGTCCAGTTACGCTGGAGTC (SEQ IDNO:861), and ACTGACCATTTAAATCATACCTGACC (SEQ ID NO:862). This PCRproduct is used for electroporation (using standard methods such asdescribed elsewhere herein) and recombination into the genome followingthe Red/ET recombination method of Gene Bridges described above. Aftertransformation positive recombinants are selected on media containingzeocin or blasticidin antibiotics. Curing of the resistance marker isaccomplished by expression of the Cre-recombittase according to standardmethods. Table 31 shows strains having genotypes that compose replacedpromoters. These are shown as “T5” followed by the affected gene(s).

Part 4: Construction of Plasmids

The following table summarizes the construction of plasmids that wereused in strains described below. To make the plasmids, a respective geneor gene region of interest was isolated by either PCR amplification andrestriction enzyme (RE) digestion or direct restriction enzyme digestionof an appropriate source carrying the gene. The isolated gene was thenligated into the desired vector, transformed into E. coli 10G (Lucigen,Middleton, Wis.) competent cells, screened by restriction mapping andconfirmed by DNA sequencing using standard molecular biology procedures(e.g., Sambrook and Russell, 2001).

It is noted that among these plasmids are those that comprisemono-functional malonyl-CoA reductase activity. Particularly, truncatedportions of malonyl-CoA reductase from C. aurantiacus were constructedby use of PCR primers adjacent, respectively, to nucleotide basesencoding amino acid residues 366 and 1220, and 496 and 1220, of thecodon-optimized malonyl-CoA reductase from pTRC-ptrc-mcr-amp. Also, amalonyl-CoA reductase from Erythrobacter sp, was incorporated intoanother plasmid. As for other plasmids, these were incorporated intostrains and evaluated as described below.

TABLE 30 Gene(s) or Vector and Catalog Cloning Method/ Plasmid PlasmidRegion Name *Supplier Number Gene(s) Source Name SEQ ID NO:Erythrobacter sp pTRCHisA V360-20 RE (NcoI/BglII)/ pTrc-ptrc- 871 MCR *ApUC 57-Eb mcr Ebmcr-amp (SEQ ID NO: 905) Truncated C. pTRCHisA V360-20PCR, RE pTrc-ptrc- 872 aurantiacus mcr *A (NcoI/HindIII)/ (366-(366-1220) pTRC-ptrc mcr- 1220)mcr- amp ptrc-ydfG- kan Truncated C.pTRCHisA V360-20 PCR, RE pTrc-ptrc- 873 aurantiacus mcr *A(NcoI/HindIII)/ ydfG-ptrc- (496-1220) pTRC-ptrc mcr- (496- amp 1220)mcr-amp mcr pTRCHisA V360-20 PCR, RE pTrc-ptrc- 874 *A (NcoI/HindIII)/mcr-amp SEQ ID No. 003 mcr pTRCHisA V360-20 RE (AhdI, pTrc-ptrc- 875 *Ablunted) for Kan mcr-kan insertion/pTRC- ptrc mcr-amp mcr/cynTS pTRCHisAV360-20 RE/(NdeI, pTrc-ptrc- 876 *A blunted: pTRC mcr-kan- ptrc-mcrkan), cynTS (EcoRV: pSMARTHC ampcynTS) accABCD pJ251 N/A RE (EcoNI,AseI, pJ251-cat- 877 *C blunted) for Cat PtpiA- insertion/SEQ ID accAD-No 820 PrpiA-accBC pntAB pACYC184 E4152S RE (NruI, PciI, pACYC184- 878cat blunted) self- cat-PtalA- *B ligate/ pntAB pACYC184-cat-PtpiA-accAD- PrpiA-accBC- ptalA-pntAB acccABCD/pntAB pACYC184 E4152SRE/(EcoRV, AvaI, pACYC184- 879 cat BseB1, blunted: cat-PtpiA- *BpACYC184), accAD- (BamHI, blunted: PrpiA- pJ244-pntAB- accBC- accABCD)ptalA-pntAB accABCD/udhA pACYC184 E4152S RE (SwaI, ApaI)/ pACYC184- 880cat pJ244-pTal-udhA cat-PtpiA- *B accAD- PrpiA- accBC- ptalA-udhAaccABCD/T5- pACYC184 E4152S RE (SwaI, NdeI)/ pACYC184- 881 udhA catpACYC184-cat- cat-PtpiA- *B PtpiA-accAD- accAD- PrpiA-accBC PrpiA- PCR,RE (PmeI, accBC-T5- NdeI)/BX_00635 udhA mcr/serA pTRCHisA V360-20 RE(PciI, blunted) pTrc-ptrc- 882 *A for pTpiA serA mcr-kan- insertion/SEQID PtpiA-serA No. 0047 fabF pTRCHisA V360-20 PCR, RE pTrc-ptrc- 883 *A(NcoI/PstI)/ fabF-amp E. coli K12 genome mcr pACYC177 E4151S PCR(blunt)/ pACYC177- 884 kan pTRC-ptrc mcr- kan-ptrc- *B amp mcrmcr/accABCD pACYC177 E4151S RE/(SwaI, XbaI: pACYC177- 885 kan pACYC 177kan kan-ptrc- *B ptrc-mcr), (PmeI, mcr-PtpiA- XbaI: pJ251-cat- accAD-PtpiA-accAD- PrpiA-accBC PrpiA-accBC *A: Invitrogen, Carlsbad, CA; *B:New England Biolabs, Ipswich, MA; *C: DNA 2.0, Menlo Park, CA

Part 5: Cloning of pACYC-cat-accABCD-P_(T5)-udhA.

The P_(ta1A) promoter driving expression of udhA in pACYC-cat-accABC-udhA was replaced with the stronger T5 promoter. The genomicP_(T5)-udhA construct from strain BX_00635—was amplified using primerAS1170 (udhA 300 bp upstream). See SEQ ID NO:886 for sequence of udhA).PCR fragments of P_(T5)-udhA obtained above were digested with PmeI andNdeI (New England BioLabs, Ipswich, Mass.). VectorpACYC-cat-accABCD-P_(ta1)-udhA was similarly digested with SwaI and NdeI(New England BioLabs). The two digested DNA fragments were ligated andtransformed to create pACYC-cat-accABCD-P_(T5)-udhA (SEQ ID NO:887).Plasmid digests were used to confirm the correct sequence. This plasmidis incorporated into strains shown in Table 31.

Part 6: Strain Construction

Using constructs made by the above methods, strains shown in Table 31,given the indicated Strain Names, were produced providing the genotypes.This is not meant to be limiting, and other strains may be made usingthese methods and following the teachings provided in this application,including providing different genes and gene regions for tolerance,and/or 3-HP production and modifications to modulate the fatty acidsynthase system. Further to the latter, such strains may be produced bychromosomal modifications and/or introduction of non-chromosomalintroductions, such as plasmids.

As to the latter, according to the respective combinations indicated inTable 38 below, the plasmids described above were introduced into therespective strains. All plasmids were introduced at the same time viaelectroporation using standard methods. Transformed cells were grown onthe appropriate media with antibiotic supplementation and colonies wereselected based on their appropriate growth on the selective media.

TABLE 31 Strain Name Strain Genotype BW25113 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568, hsdR514 BX_0591 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frtBX_0595 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1,Δ(rhaD-rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt,ΔpoxB::frt, Δpta-ack::frt, fabI^(ts)(S241F)-zeoR BX_0619 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt,fabIts (S241F)-zeoR, T5-pntAB- BSD BX_0634 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabI^(ts)(S241F)-zeoR, T5-pntAB, T5-aceEF BX_0635 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabI^(ts)(S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA-BSD BX_0636 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt,fabI^(ts) (S241F)-zeoR, T5-aceEF BX_0637 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabI^(ts)(S241F)-zeoR, T5-aceEF, T5-udhA-BSD BX_0638 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabI^(ts)(S241F)-zeoR, T5-pntAB, T5-aceEF, ΔaldB::frt BX_0639 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt,fabI^(ts) (S241F)-zeoR, T5-pntAB, T5-aceEF, ΔtrpR::kan BX_0651 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568,hsdR514, fabI^(ts) (S241F)-zeoR BX_0652 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabI^(ts)(S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA, ΔarcA::kan BX_0653 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt,fabI^(ts) (S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA, ΔpuuC::kan BX_0654F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD-rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt,fabI^(ts) (S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA, ΔaldA::kan

Example 12B Preparing a Genetically Modified E. coli Host CellComprising malonyl-CoA-reductase (Mcr) in Combination with Other GeneticModifications to Increase 3-HP Production Relative to a Control E. coliCell (Prophetic)

Genetic modifications are made to introduce a vector comprising minsBsuch as from Pseudomonas auruginos, which further is codon-optimized forE. coli. Vectors comprising galP and a native or mutated ppc also may beintroduced by methods known to those skilled in the art (see, e.g.,Sambrook and Russell, Molecular Cloning: A Laboratory Manual, ThirdEdition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., “Sambrook and Russell, 2001”), additionallyrecognizing that mutations may be made by a method using, the XL1.-Redmutator strain, using appropriate materials following a manufacturer'sinstructions (Stratagene QuikChange Mutagenesis Kit, Stratagene, LaJolla, Calif. USA) and selected for or screened under standardprotocols.

Also, genetic modifications are made to reduce or eliminate theenzymatic activities of E. coli genes as desired. These geneticmodifications are achieved by using the RED/ET homologous recombinationmethod with kits supplied by Gene Bridges (Gene Bridges GmbH, Dresden,Germany, www.genebridges.com) according to manufacturer's instructions.

Also, in some embodiments genetic modifications are made to increase theNADPH cellular pool. Non-limiting examples of some targets for geneticmodification are provided herein. These are pgi (in a mutated form),pntAB, overexpressed, gapA:gapN substitution/replacement, and disruptingor modifying a soluble transhydrogenase such as sthA, and geneticmodifications of one or more of zwf, gnd, and edd.

The so-genetically modified microorganism of any such engineeredembodiment is evaluated and found to exhibit higher productivity of 3-HPcompared with a control E. coli lacking said genetic modifications.Productivity is measured by standard metrics, such as volumetricproductivity (grams of 3-HP/hour) under similar culture conditions.

Example 12C Mutational Development of Selected Polynucleotides(Prophetic)

A selected gene sequence, such as a nucleic acid sequence that encodesfor any of SEQ ID NOs:783-791, is subjected to a mutation developmentprotocol, starting by constructing a mutant library of a native orpreviously evolved and/or codon-optimized polynucleotide by use of anerror-inducing PCR site-directed mutagenesis method.

A polynucleotide exhibiting enzymatic activity of the selected gene(which may be any disclosed herein, e.g., an aminotransferase or mmsB)is cloned into an appropriate expression system for E. coli. Thissequence may be codon optimized. Cloning of a codon-optimizedpolynucleotide and its adequate expression will be accomplished via genesynthesis supplied from a commercial supplier using standard techniques.The gene with he synthesized with an eight amino acid C-terminal tag toenable affinity based protein purification. Once obtained using standardmethodology, the gene will be cloned into an expression system usingstandard techniques.

The plasmid containing the above-described polynucleotide will bemutated by standard methods resulting in a large library of mutants(>10⁶). The mutant sequences will be excised from these plasmids andagain cloned into an expression vector, generating a final library ofgreater than 10⁶clones for subsequent screening. These numbers ensure agreater than 99% probability that the library will contain a mutation inevery amino acid encoded by sequence. It is acknowledged that eachmethod of creating a mutational library has its own biases, includingtransformation into mutator strains of E. coli, error prone PCR, and inaddition more site directed muagenesis.

In some embodiments, various methods may be considered and possiblyseveral explored in parallel. One such method is the use of the XL1-Redmutator strain, which is deficient in several repair mechanismsnecessary for accurate DNA replication and generates mutations inplasmids at a rate 5,000 times that of the wild-type mutation rate, maybe employed using appropriate materials following a nranufacturer'sinstructions (See Stratagene QuikChange Mutagenesis Kit, Stratagene, LaJolla, Calif. USA). This technique or other techniques known to thoseskilled in the art, may be employed and then a population of suchmutants, e.g., in a library, is evaluated, such as by a screening orselection method, to identify clones having a suitable or favorablemutation.

With the successful construction of a mutant library, it will bepossible to screen this library for increased activity, such asincreased malonyl-CoA reductase activity. The screening process will bedesigned to screen the entire library of greater than 10⁶ mutants. Thisis done by screening methods suited to the particular enzymaticreaction.

Example 13 Evaluation of 3-HP Production Using Strains of Example 12

3-HP production by BX3_0194 was demonstrated at 100-mL scale in SM3(minimal salts) media. Cultures were started from freezer stocks bystandard practice (Sambrook and Russell, 2001) into 50 mL, of LB mediaplus 100 μg/mL ampicillin and grown to stationary phase overnight at 37°C. with rotation at 225 rpm. Five ml of this culture were transferred to100 ml of SM3 media plus 40 g/L glucose, 100 μg/ml ampicillin, and 1 mMIPTG in triplicate 250-ml baffled flasks and incubated at 37° C., 225rpm. To monitor cell growth and 3-HP production by these cultures,samples (2 ml) were withdrawn at designated time points for opticaldensity measurements at 600 nm (OD₆₀₀, 1 cm pathlength) and pelleted bycentrifugation at 12,000 rpm for 5 min and the supernatant collected foranalysis of 3-HP production as described under “Analysis of cultures for3-HP production” in the Common Methods section. Dry cell weight (DCW) iscalculated as 0.33 times the measured OD₆₀₀ value, based on baseline DCWto OD₆₀₀ determinations. All data are the average of triplicatecultures. For comparison purposes, the specific productivity iscalculated from the averaged data at the 24-h time point and expressedas g 3-HP produced per gDCW. Under these conditions, no 3-HP is producedafter 24 hours in a culture growing to an OD₆₀₀ that corresponds toapproximately 1.0 g DCW. Production of 3-HP by strain BX3_0194 in SM3medium is shown in Table 32.

TABLE 32 Production of 3-HP by BX3_0194 in SM3 medium Time (hr) 3HP(g/L) OD₆₀₀ 4 0 1.3 6 0 2.3 8 0 2.8 24 0 3.4

Production by strain BX3_0194 in SM3 medium in the presence of 10 μg/mlcerulenin is shown in Table 33. In the presence of cerulenin, aninhibitor of the fatty acid synthase system, internal pools of themalonyl-CoA precursor are proposed to increase thus leading to increasedproduction of 3-HP. As may be seen by comparison to the results withoutcenulenin (Table 32), substantially more 3-HP is produced at every timepoint. Under these conditions, the specific productivity after 24 hoursis 1.3 g 3HP per gDCW.

TABLE 33 Production of 3-HP by BX3_0194 in SM3 medium and the presenceof 10 μg/ml cerulenin Time (hr) 3HP (g/L) OD₆₀₀ 4 0.003 1.3 6 0.14 2.6 80.43 3.1 24 1.43 3.3

3-HP production by BX3_0195 was demonstrated at 100-mL scale in SM3(minimal salts) media. Cultures were started from freezer stocks bystandard practice (Sambrook and Russell, 2001) into 50 mL, of LB mediaplus 10 μg/mL ampicillin and grown to stationary phase overnight at 37°C. with rotation at 225 rpm. Five ml of this culture were transferred to100 ml of SM3 media plus 40 g/L glucose, 100 μg/ml ampicillin, and 1 mMIPTG in triplicate 250-ml baffled flasks and incubated at 37° C., 225rpm. To monitor cell growth and 3-HP production by these cultures,samples (2 ml) were withdrawn at designated time points for opticaldensity measurements at 600 nm (OD₆₀₀, 1 cm pathlength) and pelleted bycentrifugation at 12,000 rpm for 5 min and the supernatant collected foranalysis of 3-HP production as described under “Analysis of cultures for3-HP production” in the Common Methods section. Dry cell weight (DCW) iscalculated as 0.33 times the measured OD₆₀₀ value, based on baseline DCWto OD₆₀₀ determinations. All data are the average of triplicatecultures. For comparison purposes, the specific productivity iscalculated from the averaged data at the 24-h time point and expressedas g 3-HP produced per gDCW. Under these conditions, no 3HP is producedafter 24 hours in a culture growing to and OD₆₀₀ that corresponds toapproximately 1.65 g DCW. Production of 3-HP by strain BX3_0195 in SM3medium is shown in Table 34.

TABLE 34 Production of 3-HP by BX3_0195 in SM3 medium Time (hr) 3HP(g/L) OD₆₀₀ 4 0 0.92 6 0 1.35 8 0 2.36 24 0 5.00

Production by strain BX3_0195 in SM3 medium in the presence of 10 μg/mlcerulenin is shown in Table 35. In the presence of cerulenin, aninhibitor of the fatty acid synthase system, internal pools of themalonyl-CoA precursor are proposed to increase thus leading to increasedproduction of 3-HP. As may be seen by comparison to the results withoutcerulenin (Table 34), substantially more 3-HP is produced at every timepoint. Under these conditions, the specific productivity after 24 hoursis 0.54 g 3HP per gDCW.

TABLE 35 Production of 3-HP by BX3_0195 in SM3 medium and the presenceof 10 μg/ml cerulenin Time (hr) 3HP (g/L) OD₆₀₀ 4 0.003 0.97 6 0.07 1.578 0.31 2.36 24 1.17 6.59

3-HP production by BX3_0206 was demonstrated at 100-mL scale in SM3(minimal salts) media. Cultures were started from freezer stocks bystandard practice (Sambrook and Russell, 2001) into 50 mL of LB mediaplus 35 μg/mL, kanamycin and grown to stationary phase overnight at 37°C. with rotation at 225 rpm. Five ml of this culture were transferred to100 ml of SM3 media plus 40 g/L glucose and 35 μg/ml kanamycin intriplicate 250-ml baffled flasks and incubated at 37° C., 225 rpm. Tomonitor cell growth and 3-HP production by these cultures, samples (2ml) were withdrawn at designated time points for optical densitymeasurements at 600 nm (OD₆₀₀, 1 cm pathlength) and pelleted bycentrifugation at 12,000 rpm for 5 min and the supernatant collected foranalysis of 3-HP production as described under “Analysis of cultures for3-HP production” in the Common Methods section. Dry cell weight (DCW) iscalculated as 0.33 times the measured OD₆₀₀ value, based on baseline DCWto OD₆₀₀ determinations. All data are the average of triplicatecultures. For comparison purposes, the specific productivity iscalculated from the averaged data at the 24-h time point and expressedas g 3-HP produced per gDCW. Under these conditions, the specificproductivity after 24 hours is 0.05 g 3-HP per gDCW. Production of 3-HPby strain BX3_0206 in SM3 iridium is shown in Table 36.

TABLE 36 Production of 3-HP by BX3_0206 in SM3 medium Time (hr) 3HP(g/L) OD₆₀₀ 24 0.01 6.5

Production by strain BX3_0206 in SM3 medium in the presence of 10 μg/mlcerulenin is shown in Table 37. In the presence of cerulenin, aninhibitor of the fatty acid synthase system internal pools of themalonyl-CoA precursor are proposed to increase thus leading to increasedproduction of 3-HP. As may be seen by comparison to the results withoutcerulenin (Table 36), substantially more 3-HP is produced after 24hours. Under these conditions, the specific productivity after 24 hoursis 0.20 g 3HP per gDCW, an approximately 40-fold increase relative tothe results without cerulenin.

TABLE 37 Production of 3-HP by BX3_0195 in SM3 medium and the presenceof 10 μg/ml cerulenin Time (hr) 3HP (g/L) OD₆₀₀ 24 0.43 6.4

Example 13A Evaluation of Strains for 3-HP Production

3-HP production in biocatalysts (strains) listed in the following tablewas demonstrated at 100-mL scale in SM3 (minimal salts) media. SM3 usedis described under the Common Methods Section, but was supplemented with200 mM MOPS. Cultures were started from LB plates containing antibioticsby standard practice (Sambrook and Russell, 2001) into 50 mL of TB mediaplus the appropriate antibiotic as indicated and grown to stationaryphase overnight at 30° C. with rotation at 250 rpm. Five ml of thisculture were transferred to 100 ml of SM3 media plus 30 g/L glucose,antibiotic, and 1 mM IPTG (identified as “yes” under the “Induced”column) in triplicate 250 ml baffled flasks and incubated at 30° C., 250rpm. Flasks were shifted to 37° C., 250 rpm after 4 hours. To monitorcell growth and 3-HP production by these cultures, samples (2 ml) werewithdrawn at 24 hours for optical density measurements at 600 nm (OD₆₀₀,1 cm pathlength) and pelleted by centrifugation at 14000 rpm for 5 minand the supernatant collected for analysis of 3-HP production asdescribed under “Analysis of cultures for 3-HP production” in the CommonMethods section. 3-HP titer and standard deviation is expressed as g/L.Dry cell weight (DCW) is calculated as 0.33 times the measured OD₆₀₀value, based on baseline DCW per OD₆₀₀ determinations. All data are theaverage of triplicate cultures. For comparison purposes, product to cellratio is calculated from the averaged data over 24 hours and isexpressed as g 3-HP produced per gDCW. The specific productivity iscalculated from the cell/product ratio obtained over the 20 hours ofproduction and expressed as g 3-HP produced per gDCW per hour.

TABLE 38 Average 20 Hour 24 Hour 24 Hour Standard Specific Product/Strain Name Strain Host Plasmids Induced Titer Deviation ProductivityCell Ratio BX3_0274 BW25113 1) pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001<0.001 BX3_0282 BW25113 1) pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001<0.001 2) pJ251-cat-PtpiA- accAD-PrpiA-accBC BX3_0283 BW25113 1)pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001 <0.001 2) pACYC184-cat-PtalA-pntAB BX3_0275 BW25113 1) pTrc-ptrc-mcr-kan yes <0.001 0.000<0.001 <0.001 2) pACYC184-cat- PtpiA-accAD-PrpiA- accBC-ptalA-pntABBX3_0284 BW25113 1) pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001 <0.001 2)pACYC184-cat- PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0285 BX_00591 1)pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001 <0.001 BX3_0286 BX_00591 1)pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001 <0.001 2) pJ251-cat-PtpiA-accAD-PrpiA-accBC BX3_0287 BX_00591 1) pTrc-ptrc-mcr-kan yes <0.0010.000 <0.001 <0.001 2) pACYC184-cat- PtalA-pntAB BX3_0288 BX_00591 1)pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001 <0.001 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0289 BX_00591 1)pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001 <0.001 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0239 BX_00595 1)pTrc-ptrc-mcr-kan yes 2.317 0.001 0.067 1.335 BX3_0261 BX_00595 1)pTrc-ptrc-mcr-kan yes 4.576 0.327 0.187 3.748 2) pJ251-cat-PtpiA-accAD-PrpiA-accBC BX3_0290 BX_00595 1) pTrc-ptrc-mcr-kan yes 1.706 0.3960.060 1.194 2) pACYC184-cat- PtalA-pntAB BX3_0240 BX_00595 1)pTrc-ptrc-mcr-kan yes 5.878 0.684 0.228 4.563 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0267 BX_00595 1)pTrc-ptrc-mcr-kan, yes 3.440 0.205 0.160 2.912 2)pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0253 BX_00619 1)pTrc-ptrc-mcr-kan yes 1.327 0.575 0.034 0.670 BX3_0254 BX_00619 1)pTrc-ptrc-mcr-kan yes 3.131 0.058 0.136 2.711 2)pJ251-cat-PtpiA-accAD-PrpiA-accBC BX3_0263 BX_00619 1) pTrc-ptrc-mcr-kan yes 2.376 0.7170.060 1.200 2) pACYC184-cat- PtpiA-accAD-PrpiA- accBC-ptalA-pntABBX3_0268 BX_00619 1) pTrc-ptrc-mcr-kan yes 5.555 0.265 0.240 4.809 2)pACYC184-cat- PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0279 BX_00637 1)pTrc-ptrc-mcr-kan yes 3.640 0.210 0.154 3.073 2) pJ251-cat-PtpiA-accAD-PrpiA-accBC BX3_0303 BX_00637 1) pTrc-ptrc-mcr-kan yes 2.620 0.0850.065 1.297 2) pACYC184-cat- PtalA-pntAB BX3_0281 BX_00637 1)pTrc-ptrc-mcr-kan yes 4.700 0.271 0.209 4.177 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0280 BX_00637 1)pTrc-ptrc-mcr-kan yes 4.270 0.314 0.175 3.507 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0276 BX_00635 1)pTrc-ptrc-mcr-kan yes 5.110 0.542 0.210 4.196 2) pJ251-cat-PtpiA-accAD-PrpiA-accBC BX3_0304 BX_00635 1) pTrc-ptrc-mcr-kan yes 2.430 0.1470.076 1.512 2) pACYC184-cat- PtalA-pntAB BX3_0278 BX_00635 1)pTrc-ptrc-mcr-kan yes 0.790 0.015 0.034 0.672 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0277 BX_00635 1)pTrc-ptrc-mcr-kan yes 6.340 0.580 0.260 5.207 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0296 BX_00636 1)pTrc-ptrc-mcr-kan yes 3.400 0.139 0.102 2.032 BX3_0297 BX_00636 1)pTrc-ptrc-mcr-kan yes 1.830 0.144 0.069 1.376 2) pJ251-cat-PtpiA-accAD-PrpiA-accBC BX3_0298 BX_00636 1) pTrc-ptrc-mcr-kan yes 2.670 0.0650.081 1.628 2) pACYC184-cat- PtalA-pntAB BX3_0299 BX_00636 1)pTrc-ptrc-mcr-kan yes 3.200 0.418 0.121 2.412 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0300 BX_00636 1)pTrc-ptrc-mcr-kan yes 4.930 0.638 0.184 3.671 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0291 BX_00634 1)pTrc-ptrc-mcr-kan yes 1.330 0.138 0.039 0.783 BX3_0292 BX_00634 1)pTrc-ptrc-mcr-kan yes 1.209 0.087 0.030 0.599 2) pJ251-cat-PtpiA-accAD-PrpiA-accBC BX3_0293 BX_00634 1) pTrc-ptrc-mcr-kan yes 0.269 0.0350.006 0.124 2) pACYC184-cat- PtalA-pntAB BX3_0294 BX_00634 1)pTrc-ptrc-mcr-kan yes 1.588 0.136 0.046 0.927 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0295 BX_00634 1)pTrc-ptrc-mcr-kan yes 1.054 0.048 0.028 0.552 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0302 BX_00637 1)pTrc-ptrc-mcr-kan yes 3.710 0.221 0.118 2.352 BX3_0301 BX_00635 1)pTrc-ptrc-mcr-kan yes 3.150 0.576 0.101 2.027 BX3_0305 BW25113 1)pTrc-ptrc-mcr-kan- yes 0.006 0.006 0.000 0.003 cynTS 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0306 BX_00591 1)pTrc-ptrc-mcr-kan- yes 0.035 0.035 0.001 0.014 cynTS 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0258 BX_00595 1)pTrc-ptrc-mcr-kan- yes 1.190 0.046 0.039 0.771 cynTS 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-pntAB BX3_0308 BX_00634 1)pTrc-ptrc-mcr-kan- yes 0.401 0.006 0.011 0.211 cynTS 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0310 BX_00637 1)pTrc-ptrc-mcr-kan- yes 1.450 0.072 0.045 0.897 cynTS 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0309 BX_00635 1)pTrc-ptrc-mcr-kan- yes 4.079 0.054 0.155 3.098 cynTS 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0311 BX_00638 1)pTrc-ptrc-mcr-kan yes 3.040 0.227 0.119 2.387 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0312 BX_00639 1)pTrc-ptrc-mcr-amp yes 2.850 0.071 0.152 3.030 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0352 BX_0651 1)pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001 NA BX3_0353 BX_0651 1)pTrc-ptrc-mcr-kan yes <0.001 0.000 <0.001 NA 2) pJ251-cat-PtpiA-accAD-PrpiA-accBC BX3_0313 BX_00635 1) pACYC177-kan- no 0.037 0.0090.001 0.027 ptrc-mcr BX3_0313 BX_00635 1) pACYC177-kan- yes 0.031 0.0090.001 0.023 ptrc-mcr BX3_0335 BX_00635 1) pACYC177-kan- no 0.037 0.0210.001 0.020 ptrc-mcr-PtpiA-accAD- PrpiA-accBC BX3_0335 BX_00635 1)pACYC177-kan- yes 0.037 0.021 0.001 0.020 ptrc-mcr-PtpiA-accAD-PrpiA-accBC BX3_0349 BX_00591 1) pTrc-ptrc-(366- yes 0.057 0.006 0.0010.025 1220)mcr-ptrc-ydfG- kan BX3_0350 BX_00595 1) pTrc-ptrc-(366- yes1.163 0.045 0.023 0.457 1220)mcr-ptrc-ydfG- kan BX3_0351 BX_00635 1)pTrc-ptrc-(366- yes 0.658 0.060 0.020 0.390 1220)mcr-ptrc-ydfG- kan 2)pACYC184-cat- PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0358 BX_00591 1)pTrc-ptrc-ydfG-ptrc- yes 0.040 0.000 0.001 0.015 (496-1220)mcr-ampBX3_0360 BX_00635 1) pTrc-ptrc-ydfG-ptrc- yes 4.027 0.185 0.138 2.761(496-1220)mcr-amp 2) pACYC184-cat- PtpiA-accAD-PrpiA- accBC-ptalA-udhABX3_0314 BX_00635 1) pTrc-ptrc-mcr-kan- yes 1.170 0.118 0.055 1.101PtpiA-serA 2) pACYC184-cat- PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0315BX_00591 1) pACYC177-kan- no 0.013 0.006 0.000 0.008 ptrc-mcr BX3_0316BX_00595 1) pACYC177-kan- no 0.010 0.012 0.000 0.007 ptrc-mcr BX3_0333BX_00591 1) pACYC177-kan- no 0.005 0.004 0.000 0.002ptrc-mcr-PtpiA-accAD- PrpiA-accBC BX3_0334 BX_00595 1) pACYC177-kan- no0.300 0.013 0.007 0.134 ptrc-mcr-PtpiA-accAD- PrpiA-accBC BX3_0317BX_00591 1) pACYC177-kan- no <0.001 0.000 <0.2 <0.2 ptrc-mcr 2)pTrc-ptrc-fabF-amp BX3_0317 BX_00591 1) pACYC177-kan- yes 0.033 0.0240.001 0.021 ptrc-mcr 2) pTrc-ptrc-fabF-amp BX3_0338 BX_00591 1)pACYC177-kan- no 0.010 0.005 0.000 0.004 ptrc-mcr-PtpiA-accAD-PrpiA-accBC 2) pTrc-ptrc-fabF-amp BX3_0338 BX_00591 1) pACYC177-kan- yes1.580 0.142 0.006 0.116 ptrc-mcr-PtpiA-accAD- PrpiA-accBC 2)pTrc-ptrc-fabF-amp BX3_0318 BX_00595 1) pACYC177-kan- no 0.161 0.0130.005 0.097 ptrc-mcr 2) pTrc-ptrc-fabF-amp BX3_0318 BX_00595 1)pACYC177-kan- yes 1.330 0.101 0.049 0.976 ptrc-mcr 2) pTrc-ptrc-fabF-ampBX3_0339 BX_00595 1) pACYC177-kan- no 0.083 0.015 0.007 0.149ptrc-mcr-PtpiA-accAD- PrpiA-accBC 2) pTrc-ptrc-fabF-amp BX3_0339BX_00595 1) pACYC177-kan- yes 0.010 0.009 0.000 0.007ptrc-mcr-PtpiA-accAD- PrpiA-accBC 2) pTrc-ptrc-fabF-amp BX3_0319BX_00635 1) pACYC177-kan- no 0.120 0.008 0.005 0.094 ptrc-mcr 2)pTrc-ptrc-fabF-amp BX3_0319 BX_00635 1) pACYC177-kan- yes 1.068 0.4500.043 0.854 ptrc-mcr 2) pTrc-ptrc-fabF-amp BX3_0341 BX_00635 1)pACYC177-kan- no 0.327 0.021 0.009 0.171 ptrc-mcr-PtpiA-accAD-PrpiA-accBC 2) pTrc-ptrc-fabF-amp BX3_0341 BX_00635 1) pACYC177-kan- yes0.140 0.017 0.015 0.293 ptrc-mcr-PtpiA-accAD- PrpiA-accBC 2)pTrc-ptrc-fabF-amp BX3_0342 BX_00635 1) pTrc-ptrc-mcr-kan yes 0.3410.055 0.009 0.188 2) pACYC184-cat- PtpiA-accAD-PrpiA- accBC-T5-udhABX3_0343 BX_00635 1) pTrc-ptrc-mcr-kan- yes 1.927 0.047 0.077 1.536cynTS 2) pACYC184-cat- PtpiA-accAD-PrpiA- accBC-T5-udhA BX3_0344BX_00652 1) pTrc-ptrc-mcr-amp yes 1.562 0.280 0.040 0.797 BX3_0345BX_00652 1) pTrc-ptrc-mcr-amp yes 5.195 0.229 0.184 3.678 2)pJ251-cat-PtpiA- accAD-PrpiA-accBC BX3_0346 BX_00652 1)pTrc-ptrc-mcr-amp yes 1.781 0.132 0.056 1.119 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0347 BX_00653 1)pTrc-ptrc-mcr-amp yes 1.370 0.307 0.049 0.977 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0348 BX_00654 1)pTrc-ptrc-mcr-amp yes 1.387 0.184 0.049 0.982 2) pACYC184-cat-PtpiA-accAD-PrpiA- accBC-ptalA-udhA BX3_0324 BX_00591 1)pTrc-ptrc-Ebmcr- yes 0.009 0.002 0.000 0.004 amp BX3_0328 BX_00595 1)pTrc-ptrc-Ebmcr- yes 0.011 0.005 0.000 0.006 amp

Example 13B Evaluation of BX3_240 Strain with Carbonate Addition

3-HP production in E. coli BX3_240 (made by methods above) was evaluatedat 100-mL scale in SM3 (minimal salts) media having added sodiumcarbonate. SM3 used is described under the Common Methods Section, towhich was added 10 mM, 20 mM and 50 mM Na₂CO₃ as treatments. Cultureswere started from LB plates containing antibiotics by standard practice(Sambrook and Russell, 2001) into 50 mL of TB media plus the appropriateantibiotics kan and cat and grown to stationary phase overnight at 30°C. with rotation at 250 rpm. Five ml of this culture were transferred to100 ml of SM3 media plus 30 g/L glucose, antibiotic, the indicatedsodium carbonate. 0.1% yeast extract and 1 mM IPTG in triplicate 250-mlbaffled flasks and incubated at 30° C., 250 rpm. Flasks were shifted to37° C., 250 rpm after 4 hours. To monitor cell growth and 3-HPproduction by these cultures, samples (2 ml) were withdrawn at 24, 48and 60 hours for optical density measurements at 600 nm. (OD600, 1 cmpath length) and pelleted by centrifugation at 14000 rpm for 5 min andthe supernatant collected for analysis of 3-HP production as describedunder “Analysis of cultures for 3-HP production” in the Common Methodssection. 3-HP titer and standard deviation is expressed as g/L. Dry cellweight (DCW) is calculated as 0.33 times the measured OD600 value, basedon baseline DCW per OD600 determinations. All data are the average oftriplicate cultures. For comparison purposes, product to cell ratio iscalculated from the averaged data over 60 hours and is expressed as g3-HP produced per gDCW.

3-HP titer were 0.32 (+/−0.03), 0.87 (+/−0.10), 2.24 (+/−0.03), 4.15(+/−0.27), 6.24 (+/−0.51), 7.50 (+/−0.55) and 8.03 (+/−0.14) g/L at 9,11, 15, 19, 24, 48 and 60 hr, respectively. Biomass concentrations were0,54 (+/−0.02), 0.79 (+/−0.03), 1.03 (+/−0.06), 1.18 (+/−0.04), 1.20(+/−0.12), 1.74 (+/−0.30) and 1.84 (+/−0.22) at 9, 11, 15, 19, 24, 48and 60 hr, respectively. Maximum product to cell ratio was 4.6 g 3-HP/gDCW.

Example 14 General Example of Genetic Modification to a Host Cell(Prophetic and Non-Specific)

In addition to the above specific examples, this example is meant todescribe a non-limiting approach to genetic modification of a selectedmicroorganism to introduce a nucleic acid sequence of interest.Alternatives and variations are provided within this general example.The methods of this example are conducted to achieve a combination ofdesired genetic modifications in a selected microorganism species, suchas a combination of genetic modifications as described in sectionsherein, and their functional equivalents, such as in other bacterial andother microorganism species.

A gene or other nucleic acid sequence segment of interest is identifiedin a particular species (such as E. coli as described herein) and anucleic acid sequence comprising that gene or segment is obtained.

Based on the nucleic acid sequences at the ends of or adjacent the endsof the segment of interest, 5′ and 3′ nucleic acid primers are prepared.Each primer is designed to have a sufficient overlap section thathybridizes with such ends or adjacent regions. Such primers may includeenzyme recognition sites for restriction digest of transposase insertionthat could be used for subsequent vector incorporation or genomicinsertion. These sites are typically designed to be outward of thehybridizing overlap sections. Numerous contract services are known thatprepare primer sequences to order (e.g., Integrated DNA Technologies,Coralville, Iowa USA).

Once primers are designed and prepared, polymerase chain reaction (PCR)is conducted to specifically amplify the desired segment of interest.This method results in multiple copies of the region of interestseparated from the microorganism's genome. The microorganism's DNA, theprimers, and a thermophilic polymerase are combined in a buffer solutionwith potassium and divalent cations (e.g., Mg or Mn) and with sufficientquantities of deoxynucleoside triphosphate molecules. This mixture isexposed to a standard regimen of temperature increases and decreases.However, temperatures, components, concentrations, and cycle times nayvary according to the reaction according to length of the sequence to becopied, annealing temperature approximations and other factors known orreadily learned through routine experimentation by one skilled in theart.

In an alternative embodiment the segment of interest may be synthesized,such as by a commercial vendor, and prepared via PCR, rather thanobtaining from a microorganism or other natural source of DNA.

The nucleic acid sequences then are purified and separated, such as onan agarose gel via electrophoresis. Optionally, once the region ispurified it can be validated by standard DNA sequencing methodology andmay be introduced into a vector. Any of a number of vectors may be used,which generally comprise markers known to those skilled in the art, andstandard methodologies are routinely employed for such introduction.Commonly used vector systems are pSMART (Lucigen, Middleton, Wis.), pETE. coli EXPRESSION SYSTEM (Stratagene, La Jolla, Calif.), pSC-BStrataClone Vector (Stratagene, La Jolla, Calif.), pRANGER-BTB vectors(Lucigen, Middleton, Wis.), and TOPO vector (Invitrogen Corp, Carlsbad,Calif., USA). Similarly, the vector then is introduced into any of anumber of host cells. Commonly used host cells are E. cloni 10G(Lucigen, Middleton, Wis.), E. cunni 10GF′ (Lucigen, Middleton, Wis.),StrataClone Competent cells (Stratagene, La Jolla, Calif.), E. coliBL21, E. coli BW25113, and E. coli K12 MG1655. Some of these vectorspossess promoters, such as inducible promoters, adjacent the region intowhich the sequence of interest is inserted (such as into a multiplecloning site), white other vectors, such as pSMART vectors (Lucigen,Middleton, Wis.), are provided without promoters and withdephosporylated blunt ends. The culturing of such plasmid-laden cellspermits plasmid replication and thus replication of the segment ofinterest, which often corresponds to expression of the segment ofinterest.

Various vector systems comprise a selectable marker, such as anexpressible gene encoding a protein needed for growth or survival underdefined conditions. Common selectable markers contained on backbonevector sequences include genes that encode for one or more proteinsrequired for antibiotic resistance as well as genes required tocomplement auxotrophic deficiencies or supply critical nutrients notpresent or available in a particular culture media. Vectors alsocomprise a replication system suitable for a host cell of interest.

The plasmids containing the segment of interest can then be isolated byroutine methods and are available for introduction into othermicroorganism host cells of interest. Various methods of introductionare known in the art and can include vector introduction or genomicintegration. In various alternative embodiments the DNA segment ofinterest may be separated from other plasmid DNA if the former will beintroduced into a host cell of interest by means other than suchplasmid.

While steps of the general prophetic example involve use of plasmids,other vectors known in the art may be used instead. These includecosmids, viruses (e.g., bacteriophage, animal viruses, plant viruses),and artificial chromosomes (e.g., yeast artificial chromosomes (YAC) andbacteria artificial chromosomes (BAC)).

Host cells into which the segment of interest is introduced may beevaluated for performance as to a particular enzymatic step, and/ortolerance or bio-production of a chemical compound of interest.Selections of better performing genetically modified host cells may bemade, selecting for overall performance, tolerance, or production oraccumulation of the chemical of interest.

It is noted that this procedure may incorporate a nucleic acid sequencefor a single gene (or other nucleic acid sequence segment of interest),or multiple genes (under control of separate promoters or a singlepromoter), and the procedure may be repeated to create the desiredheterologous nucleic acid sequences in expression vectors, which arethen supplied to a selected microorganism so as to have, for example, adesired complement of enzymatic conversion step functionality for any ofthe herein-disclosed metabolic pathways. However, it is noted thatalthough many approaches rely on expression via transcription of all orpart of the sequence of interest, and then translation of thetranscribed mRNA to yield a polypeptide such as an enzyme, certainsequences of interest may exert an effect by means other than suchexpression.

The specific laboratory methods used for these approaches are well-knownin the art and may be found in various references known to those skilledin the art, such as Sambrook and Russell, Molecular Cloning: ALaboratory Manual, Third Edition 2001 (volumes 1-3), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (hereinafter, Sambrook andRussell, 2001).

As an alternative to the above, other genetic modifications may also bepracticed, such as a deletion of a nucleic acid sequence of the hostcell's genome. One non-limiting method to achieve this is by use ofRed/ET recombination, known to those of ordinary skill in the art anddescribed in U.S. Pat. Nos. 6,355,412 and 6,509,156, issued to Stewartet al. and incorporated by reference herein for its teachings of thismethod. Material and kits for such method are available from GeneBridges (Gene Bridges GmbH, Dresden, Germany, <<www.genebridges.com>>),and the method may proceed by following the manufacturer's instructions.Targeted deletion of genomic DNA may be practiced to alter a host cell'smetabolism so as to reduce or eliminate production of undesiredmetabolic products. This may be used in combination with other geneticmodifications such as described herein in this general example.

Example14A Utilization of Sucrose as the Feedstock for Production of3-HP and Other Products (Partial Prophetic)

Common laboratory and industrial strains of E. coli, such as the strainsdescribed herein, are not capable of utilizing sucrose as the solecarbon source, although this property is found in a number of wildstrains, including pathogenic E. coli strains. Sucrose, andsucrose-containing feedstocks such as molasses, are abundant and oftenused as feedstocks for the production by microbial fermentation oforganic acids, amino acids, vitamins, and other products. Thus furtherderivatives of the 3-HP-producing strains that are capable of utilizingsucrose would expand the range of feedstocks that can be utilized toproduce 3-HP.

Various sucrose uptake and metabolism systems are known in the art (forexample, U.S. Pat. No. 6,960,455), incorporated by reference for suchteachings. We describe the construction of E. coli strains that harborthe csc genes confering the ability to utilize sucrose via anon-phosphotransferase system, wherein the csc genes constitute cscA,encoding a sucrose hydrolase, cscB, encoding a sucrose permease, cscK,encoding a fructokinase, and cscR, encoding a repressor. The sequencesof these genes are annotated in the NCBI database as accession No.X81461 AF473544. To allow efficient expression utilizing codons that arehighly abundant in E. coli genes, an operon containing cscB, cscK, andcscA was designed and synthesized using the services of a commercialsynthetic DNA provider (DNA 2.0, Menlo Park, Calif.). The amino acidsequences of the genes are set forth as, respectively, cscB—SEQ. ID. No.888; cscA—SEQ. ID. No. 889; csck—SEQ. ID. No. 890. The synthetic operonconsisted of 60 base pairs of the region of the E. coli genomeimmediately 5′ (upstream) of the adhE gene, a consensus strong promoterto drive expression of the csc genes, the coding regions for cscB, cscK,and cscA with short intergenic regions containing ribosome binding sitesbut no promoters, and 60 bp immediately 3′ (downstream) of the adhEgene. The segments homologous to sequences flanking the adhE gene willbe used to target insertion of the car operon genes into the E. colichromosome, with the concomittent deletion of adhE. The nucleotidesequence of the entire synthetic construct is shown as SEQ. ID. No. 891.The synthetic ccs operon is constructed in plasmid pJ214 (DNA 2.0, MenloPark, Calif.) that provides an origin of replication derived fromplasmid pl5A and a gene conferring resistance to ampicillin. Thisplasmid is denoted pSUCR. A suitable host cell, such as E. coli strainBX_595, is transformed simultaneously with pSUCR and with plasmidpTrc_kan_mcr or other suitable plasmid, and transformed strains selectedfor on LB medium plates containing ampicillin and kanamycin.Transformants carrying both plasmids are grown and evaluated for 3-HPproduction in shake flasks as described in Example 13, except that theglucose in SM3 medium is replaced with an equal concentration ofsucrose.

Genes that confer functions to enable utilization of sucrose by E. colican also be obtained from the natural isolate pUR400 (Cowan, P. J., etal. J. Bacteriol. 173;7464-7470, 1991) which carries genes for thephosphoenolpyruvate-dependent carbohydrate uptake phosphotransferasesystem (PTS). These genes consist of scrA, encoding the enzyme IIcomponent of the PTS transport complex, serB, encoding sucrose-6phosphate hydrolase, scrK, encoding fructokinase, and scrY, encoding aporin. These genes may be isolated or synthesized as described above,incorporated on a plasmid, and transformed into a suitable host cell,such as E. coli strain BX_595, simultaneously with plasmid pTrc_kan_mcror other suitable plasmid, and transformed strains selected for on LBmedium plates containing the appropriate antibiotics. Transformantscarrying both plasmids are grown and evaluated for 3-HP production inshake flasks as described in Example 13, except that the glucose in SM3medium is replaced with an equal concentration of sucrose.

Example 14B Construction and Evaluation of Additional Strains(Prophetic)

Other strains are produced that comprise various combinations of thegenetic elements (additions, deletions and modifications) describedherein are evaluated for and used for 3-HP production, includingcommercial-scale production. The following table illustrates a number ofthese strains.

Additionally, a further deletion or other modification to reduceenzymatic activity, of multifunctional 2-keto-3-deoxygluconate6-phosphate aldolase and 2-keto-4-hydroxyglutarate aldolase andoxaloacetate decarboxylase (eda in E. coli), may be provided to variousstrains. Further to the latter, in various embodiments combined withsuch reduction of enzymatic activity of multifunctional2-keto-3-deoxygluconate 6-phosphate aldolase and2-keto-4-hydroxyglutarate aldolase and oxaloacetate decarboxylase (edain E. coli), further genetic modifications may be made to increase aglucose transporter (e.g. galP in E. coli) and/or to decrease activityof one or more of heat stable, histidyl phosphorylatable protein (ofPTS) (ptsH (HPr) in E. coli), phosphoryl transfer protein (of PTS) (ptsIin E. coli), and the polypeptide chain of PTS (Crr in E. coli).

These strains are evaluated in either flasks, or fermentors, using themethods described above. Also, it is noted that after a given extent ofevaluation of strains that comprise introduced plasmids, the geneticelements in the plasmids may be introduced into the microorganismgenome, such as by methods described herein as well as other methodsknown to those skilled in the art.

TABLE 39 Strain Host Plasmids BX3P_001 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr rhaB)568, hsdR514,ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, fabB-tS BX3P_002 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr,rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt,2)accABCD Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF, T5-pntAB,T5-udhA, fabB-tS BX3P_003 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3),LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCD- Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, udhA fabB-tS BX3P_004 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcrrhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt,Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, relA,spoT BX3P_005 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1,Δ(rhaD- 1) ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt,ΔmgsA::frt, ΔpoxB::frt, 2)accABCD Δpta-ack::frt, fabIts (S241F)-zeoR T5aceEF, T5-pntAB, T5-udhA, relA, spoT BX3P_006 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCD-Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, udhArelA, spoT BX3P_007 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-,rph-1, Δ(rhaD- 1) ptrc-mcr rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt,ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF,del-arcA:kan BX3P_008 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-,rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt,ΔmgsA::frt, ΔpoxB::frt, 2)accABCD Δpta-ack::frt, fabIts (S241F)-zeoR T5aceEF, del-arcA:kan BX3P_009 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3),LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCD- Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, del-arcA:kan udhA BX3P_010 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr rhaB)568, hsdR514,ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, del-aldA, del puuC, del arcA,del aldB, spoT, relA, T5-cynTS BX3P_011 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCDΔpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA,del-aldA, del puuC, del arcA, del aldB, spoT, relA, T5-cynTS BX3P_012F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1)ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt,ΔpoxB::frt, 2)accABCD- Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF,T5-pntAB, T5-udhA, udhA del-aldA, del puuC, del arcA, del aldB, spoT,relA, T5-cynTS BX3P_013 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-,rph-1, Δ(rhaD- 1) ptrc-mcr rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt,ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF,T5-pntAB, T5-udhA, del-aldA, del puuC, del arcA, del aldB, spoT, relA,T5-cynTS, fabB- ts BX3P_014 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3),LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCD Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, del-aldA, del puuC, del arcA,del aldB, spoT, relA, T5-cynTS, fabB- ts BX3P_015 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCD-Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, udhAdel-aldA, del puuC, del arcA, del aldB, spoT, relA, T5-cynTS, fabB- tsBX3P_016 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1,Δ(rhaD- 1) ptrc-mcr rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt,ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF,T5-pntAB, T5-udhA, T5-cynTS BX3P_017 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCDΔpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, T5-cynTSBX3P_018 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1,Δ(rhaD- 1) ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt,ΔmgsA::frt, ΔpoxB::frt, 2)accABCD- Δpta-ack::frt, fabIts (S241F)-zeoR T5aceEF, T5-pntAB, T5-udhA, udhA T5-cynTS BX3P_019 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr rhaB)568, hsdR514,ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, del puuC, del arcA, del aldB,spoT, relA, T5-cynTS BX3P_020 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3),LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCD Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, del puuC, del arcA, del aldB,spoT, relA, T5-cynTS BX3P_021 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3),LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCD- Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, udhA del puuC, del arcA, delaldB, spoT, relA, T5-cynTS BX3P_022 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr rhaB)568, hsdR514,ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, Δpta-ack::frt, fabIts(S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA, del-aldA, del puuC, del aldB,spoT, relA, T5-cynTS, fabB-ts BX3P_023 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr, rhaB)568,hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, 2)accABCDΔpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF, T5-pntAB, T5-udhA,del-aldA, del puuC, del aldB, spoT, relA, T5-cynTS, fabB-ts BX3P_024 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) ptrc-mcr,rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt,2)accABCD- Δpta-ack::frt, fabIts (S241F)-zeoR T5 aceEF, T5-pntAB,T5-udhA, udhA del-aldA, del puuC, del aldB, spoT, relA, T5-cynTS,fabB-ts BX3P_025 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1,Δ(rhaD- 1) pACYC- rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt,ΔpoxB::frt, mcr- Δpta-ack::frt, fabIts (S241F)-zeoR, T5-pntAB, T5-aceEF,T5-udhA- accABCD, 2) BSD pKK223- metE C645A BX3P_026 F-,Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) pACYC-rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, mcr-Δpta-ack::frt, fabIts (S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA-accABCD, 2) BSD pKK223-ct his-thrA BX3P_027 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) pACYC- rhaB)568, hsdR514,ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, mcr- Δpta-ack::frt,fabIts (S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA- accABCD, 2) BSDpKK223- aroH*457 BX3P_028 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3),LAM-, rph-1, Δ(rhaD- 1) pACYC- rhaB)568, hsdR514, ΔldhA::frt,ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, mcr- Δpta-ack::frt, fabIts(S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA- accABCD, 2) BSD psmart-hcamp-cadA BX3P_029 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-,rph-1, Δ(rhaD- 1) pACYC- rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt,ΔmgsA::frt, ΔpoxB::frt, mcr- Δpta-ack::frt, fabIts (S241F)-zeoR,T5-pntAB, T5-aceEF, T5-udhA- accABCD, 2) BSD psmart- hcamp-metC BX3P_030F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) pACYC-rhaB)568, hsdR514, ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, mcr-Δpta-ack::frt, fabIts (S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA-accABCD, 2) BSD psmart- hcamp- nrdAB BX3P_031 F-, Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), LAM-, rph-1, Δ(rhaD- 1) pACYC- rhaB)568, hsdR514,ΔldhA::frt, ΔpflB::frt, ΔmgsA::frt, ΔpoxB::frt, mcr- Δpta-ack::frt,fabIts (S241F)-zeoR, T5-pntAB, T5-aceEF, T5-udhA- accABCD, 2) BSDpsmart- hcamp-prs

Example 15 Prophetic Example of 3-HP Production

An inoculum of a genetically modified microorganism that possesses a3-HP production pathway and other genetic modifications as describedabove is provided to a culture vessel to which also is provided a liquidmedia comprising nutrients at concentrations sufficient for a desiredbio-process culture period.

The final broth (comprising microorganism cells, largely ‘spent’ mediaand 3-HP, the latter at concentrations, in various embodiments,exceeding 1, 2, 5, 10, 30, 50, 75 or 100 grams/liter) is collected andsubjected to separation and purification steps so that 3-HP is obtainedin a relatively purified state. Separation and purification steps mayproceed by any of a number of approaches combining variousmethodologies, which may include centrifugation, concentration,filtration, reduced pressure evaporation, liquid/liquid phase separation(including after forming a polyamine-3-HP complex, such as with atertiary amine such as CAS#68814-95-9, Alamine® 336, a triC8-10 alkylamine (Cook, Cincinnati, Ohio or Henkel Corp.), membranes, distillation,and/or other methodologies recited in this patent application,incorporated herein. Principles and details of standard separation andpurification steps are known in the art, for example in “BioseparationsScience and Engineering,” Roger G. Harrison et al., Oxford UniversityPress (2003), and Membrane Separations in the Recovery of Biofuels andBiochemicals—An Update Review, Stephen A. Leeper, pp. 99-194, inSeparation and Purification Technology, Norman N. Li and Joseph M. Calo,Eds., Marcel Dekker (1992), incorporated herein for such teachings. Theparticular combination of methodologies is selected from those describedherein, and in part is based on the concentration of 3-HP and othercomponents in the final broth.

Example 16 Prophetic Example of Conversion of 3-HP to SpecifiedDownstream Chemicals

3-HP such as from Example 13 is converted to any one or more ofpropriolactone via a ring-forming internal esterification reaction(eliminating a water molecule), ethyl-3-HP via esterification withethanol, malonic acid via an oxidation reaction, and 1,3-propanediol viaa reduction reaction.

These conversions proceed such as by organic synthesis reactions knownto those skilled in the art. Any of these conversions of 3-HP mayproceed via a chemical synthesis reaction under controlled conditions toattain a high conversion rate and yield with acceptably low by-productformation.

Example 17 Prophetic Example of Bio-Acrylic Acid Production from 3-HP

3-HP is obtained in a relatively pure state from a microbialbio-production event, such as is described in Example 15. The 3-HP isconverted to acrylic acid by a dehydration reaction, such as by heatingunder vacuum in the presence of a catalyst. More particularly, anaqueous solution of 3-HP as an acid or salt is added to a rotatableflask with a catalyst selected from Table 8, incorporated into thisexample from Section X1 above.

The temperature is raised to between 100 and 190° C. while underrotation and vacuum, with vapors collected at a condenser. Acrylic acidis collected as condensate and quantified such as by analytic proceduresdescribed herein. Various combinations of parameters, such astemperature, rate of change of temperature, purity of 3-HP solutionderived from the microbial bin production event, reduced pressure (andrate of change of pressure), and type and concentration of one or morecatalysts, are evaluated with objectives of high conversion rate withoutundesired side reactions, which might, in some production scenarios,include undesired polymerization of acrylic acid.

Example 18 Alternative Prophetic Example of Bio-Acrylic, Acid Productionfrom 3-HP

3-HP is obtained in a relatively pure state from a microbialbio-production event, such as is described in Example 15. The 3-HP isconverted to acrylic acid by a dehydration reaction, such as by heatingunder vacuum in the presence of a catalyst, however under conditionsfavoring a controlled polymerization of acrylic acid after its formationfrom 3-HP. Various combinations of parameters, such as temperature, rateof change of temperature, including removal of heat generated duringreaction, purity of 3-HP solution derived from the microbialbio-production event, reduced pressure (and rate of change of pressure),and type and concentration of one or more catalysts and/or exposure tolight, are evaluated with objectives of high conversion rate withoutundesired side reactions. Acrylic acid so formed may be separated andpurified by methods known in the art, such as those methods disclosed,supra.

Example 19 Prophetic Example of Conversions of Acrylic Acid DownstreamProducts

The acrylic acid of Example 17 is further converted to one (or more) ofthe downstream products as described herein. For example, the conversionmethod is esterification with methanol to produce methyl acrylate, orother esterifications with other alcohols for other acrylate esters,amidation to produce acrylamide, adding a nitrile moiety to produceacrylonitrile. Other additions are made as desired to obtain substituteddownstream compounds as described herein.

Example 20 Prophetic Example of Conversion of Acrylic Acid toPolyacrylic Acid

The acrylic acid of Example 17 is further converted to a polyacrylicacid by heating the acrylic acid in an aqueous solution and initiating apolymerization reaction by exposing the solution to light, andthereafter controlling the temperature and reaction rate by removingheat of the polymerization.

The specific methods and teachings of the specification, and/or citedreferences that are incorporated by reference, may be incorporated intothe above examples. Also, production of 3-HP, or one of its downstreamproducts such as described herein, may reach at least 1, at least 2, atleast 5, at least 10, at least 20, at least 30, at least 40, and atleast 50 g/liter titer in various embodiments.

Example 21 Separation and Reactive Extraction of 3-HP from FermentationBroth

A fermentation broth obtained from a 10-liter fermentor at theconclusion of a fermentation experiment was heated to 60° C. for onehour as a microorganism kill step, then adjusted to approximately 100grams per liter of 3-HP (produced by the method described in CommonMethods Section, Subsection IIIa), and pH-adjusted to approximately 7.0with ammonium sulfate. Calcium chloride at 1 M was added as a flocculentto reach a final concentration of about 8.2 g/L. Thereafter the pH wasadjusted to a pH of approximately 2.0 using sulfuric acid. Thereafter avolume of this modified fermentation broth was centrifuged atapproximately 3,200 g for 5 minutes to yield a clarified broth and apellet, which was discarded.

Portions of the clarified broth were then subjected to reactiveextraction by mixing with a tertiary amine non-polar phase comprisingvarious co-solvents. After mixing, aqueous and amine non-polar phaseswere allowed to separate, and the amine non-polar phase was removed fromthe aqueous phase, which was subjected to analysis for 3-HPconcentration by HPLC (see method in Common Methods Section). Aminesincluded Alamine 336, described above, and tripentylamine. Table 40provides a summary of the single pass extraction efficiency into therespective amine non-polar phase solutions, each respectively calculatedbased on the difference between the starting 3-HP in the portion and the3-HP in the raffinate (aqueous phase after extraction).

TABLE 40 Tripentylamine with indicated co-solvent: Alamine 336 Methylethyl Methyl tert- Butanol Butanol p-Xylene ketone butyl ether 1 2 3 4 5start mass G 10.56 21.18 21.19 21.15 21.10 Density g/mL 1.06 1.06 1.061.06 1.06 3-HP concentration start g/L 96.85 96.85 96.85 96.85 96.853-HP mass start G 0.97 1.94 1.94 1.94 1.94 mass cosolvent added G 8.5310.18 10.85 10.13 9.27 cosolvent density g/mL 0.80 0.80 0.85 0.79 0.72mass amine added G 8.51 9.90 9.93 9.90 9.82 amine density g/mL 0.80 0.770.78 0.77 0.77 mass total extractant G 17.04 20.08 20.78 20.03 19.09raffinate collected G 7.64 15.92 20.04 12.66 19.32 raffinate densityg/mL 1.08 1.07 1.07 1.06 1.08 3-HP concentration g/L 53.94 54.17 78.6952.75 67.64 raffinate 3-HP mass raffinate G 0.38 0.81 1.47 0.63 1.21 %3-HP raffinate % 0.39 0.42 0.76 0.33 0.62 extractant collected G 19.2224.40 21.26 28.13 20.07 3-HP mass extractant G 0.59 1.13 0.46 1.31 0.73% 3-HP extracted % 0.61 0.58 0.24 0.67 0.38 Total Total 3HP extracted G0.59 1.13 0.46 1.31 0.73 % 3HP extracted % 60.60 58.39 23.91 67.47 37.53

It was noted that there was substantially more emulsion formation withthe Alamine 336, and the phase separation was slower, than with thetripentylamine treatments. Nonetheless, both of these tertiary aminesdemonstrated that 3-HP would extract from the aqueous phase into thenon-polar phase (i.e., the tertiary amine with co-solvents). Theco-solvents used in this example are not meant to be limiting; otherco-solvents may be considered, e.g., pentanol, hexanol, heptanol,octanol, nonanol, decanol. Also, it is noted that hexane was tested as aco-solvent with tripentylamine but the data was not considered valid asthis sample caused a peak shift in the HPLC analysis.

Further, as described elsewhere in this application and as generallyknown in the art, there are other approaches to separation, extraction,and purification of 3-HP from a fermentation broth. Accordingly, thisexample is not meant to be limiting.

An example of recovery of the 3-HP from the non-polar phase tertiaryamine solution by back-extraction is provided in Example 22.

Example 22 3-HP Dehydration to Acrylic Acid with Acid Catalyst

Approximately 15 mL of an aqueous solution comprising about 350 grams of3-HP per liter (produced by the method described in Common MethodsSection, Subsection IIIa) was combined in a flask with approximately 15mL, of concentrated sulfuric acid. The flask was attached to a rotaryevaporator apparatus (Rotovapor Model R-210, BUCHI Labortechnik AG,Switzerland), heated in a heating bath (BUCCHI, Model B-491) to 80° C.under reduced pressure (10 to 20 mbar), and the condensate was collectedbelow a condensing apparatus operated with chilled water as the coolant.After approximately 5 hours the condensate was collected, its volumemeasured, and an aliquot submitted for HPLC analysis (see Common MethodsSection). An aliquot of the reaction mixture in the flask also wassubmitted for HPLC analysis. The HPLC analysis indicated thatapproximately 24 grams per liter of acrylic acid was obtained in thecondensate, whereas approximately 4.5 grams per liter remained in thereaction mixture of the flask. Thus, 3-HP was shown to form acrylic acidunder these conditions. This example is not meant to be limiting.

Example 23 Prophetic Example of Conversion of Acrylic Acid toPolyacrylic Acid

Acrylic acid, such as that provided in Example 22, is further convertedto a polyacrylic acid by heating the acrylic acid in an aqueous solutionand initiating a free-radical polymerization reaction by exposing thesolution to light, and thereafter controlling the temperature andreaction rate by removing heat of the polymerization.

Batch polymerization is utilized, wherein acrylic acid is dissolved inwater at a concentration of about 50 wt %. The monomer solution isdeoxygenated by bubbling nitrogen through the solution. A free-radicalinitiator, such as an organic peroxide, is optionally added (to assistthe initiation via the light source) and the temperature is brought toabout 60° C. to start polymerization.

The molecular mass and molecular mass distribution of the polymer aremeasured. Optionally, other polymer properties including density,viscosity, melting temperature, and glass-transition temperature aredetermined.

The specific methods and teachings of the specification, and/or citedreferences that are incorporated by reference, may be incorporated intothe above examples. Also, production of 3-HP, or one of its downstreamproducts such as described herein, may reach at least 1, at least 2, atleast 5, at least 10, at least 20, at least 30, at least 40, and atleast 50 g/liter titer in various embodiments.

Example 24 Prophetic Example of Bulk Polymerization of Acrylic Acid toPolyacrylic Acid

Acrylic acid, such as that provided in Example 22, is further convertedto a polyacrylic acid by bulk polymerization. Acrylic acid monomer,monomer-soluble initiators, and neutralizing base are combined in apolymerization reactor. Polymerization is initiated, and temperature iscontrolled to attain a desired conversion level. Initiators arewell-known in the art and include a range of organic peroxides and othercompounds, such as discussed above. The acrylic acid or polyacrylic acidis at least partially neutralized with a base such as sodium hydroxide.

The molecular mass and molecular mass distribution of the polymer aremeasured. Optionally, other polymer properties including density,viscosity, melting temperature, and glass-transition temperature aredetermined.

The polyacrylic acid produced is intended for use as a superabsorbentpolymer, as an absorbent for water and aqueous solutions for diapers,adult incontinence products, feminine hygiene products, and similarconsumer products, as well as for possible uses in agriculture,horticulture, and other fields.

Example 25 Prophetic Example of Production of a Superabsorbent Polymer

Acrylic acid, such as that provided in Example 22, is further convertedto a superabsorbent polyacrylic acid by solution polymerization. Anaqueous solution of acrylic acid monomer (at about 25-30 wt %),initiators, neutralizing base, antioxidants, crosslinkers (such astrimethylolpropane triacrylate) and optionally other additives arecombined in a polymerization reactor and polymerization is initiated.Bases that can be used for neutralization include but are not limited tosodium carbonate, sodium hydroxide, and potassium hydroxide.

The reactor contents are deoxygenated for 60 minutes. The temperature ofthe polymerization reaction is allowed to rise to an initial desiredlevel. The reactor is then maintained at a desired hold temperature fora period of time necessary for the desired monomer conversion to beachieved. The resulting reaction product is in the form of ahigh-viscosity gel. The high-viscosity, gel-like reaction product isthen processed into a film or a strand, dried and ground into particleswhich are screened or classified into various particle size fractions.After the polymer is dried and ground to final particulate size, it isanalyzed for residual acrylic acid and other chemicals, extractablecentrifuge capacity, shear modulus, and absorption under load. Otherpolymer properties may be measured, including molecular mass, molecularmass distribution, density, viscosity, melting temperature, and glasstransition temperature. Surface treatments may be performed by adding acrosslinking co-monomer to the surface of the polymer particles.

The polyacrylic acid produced is intended for use as a superabsorbentpolymer, as an absorbent for water and aqueous solutions for diapers,adult incontinence products, feminine hygiene products, and similarconsumer products, as well as for possible uses in agriculture,horticulture. and other fields.

Example 26 Alternative Prophetic Example of Production of aSuperabsorbent Polymer

Acrylic acid, such as that provided in Example 22, is further convertedto a superabsorbent polyacrylic acid by suspension polymerization. Anaqueous phase comprising water, acrylic acid monomer, and neutralizingbase is combined with an an oil phase comprising an inert hydrophobicliquid and optionally a suspending agent is further provided. Theaqueous phase and the oil phase are contacted under conditions(including a temperature of about 75° C.) such that fine monomerdroplets are formed. Polymerization is initiated, and the polymerizedmicroparticles of polyacrylic acid are recovered from the suspensionusing a centrifuge.

The polyacrylic acid is then dried and ground into particles which arescreened or classified into various particle size fractions. After thepolymer is dried and ground to final particulate size, it is analyzedfor residual acrylic acid and other chemicals, extractable centrifugecapacity, shear modulus, and absorption under load. Other polymerproperties may be measured, including molecular mass, molecular massdistribution, density, viscosity, melting temperature, andglass-transition temperature.

The polyacrylic acid produced is intended for use as a superabsorbentpolymer, as an absorbent for water and aqueous solutions for diapers,adult incontinence products, feminine hygiene products, and similarconsumer products, as well as for possible uses in agriculture,horticulture, and other fields.

Example 27 Prophetic Example of Conversion of Acrylic Acid to MethylAcrylate

Acrylic acid, such as that provided in Example 22, is converted tomethyl acrylate by direct, catalyzed esterification. Acrylic acid iscontacted with methanol, and the mixture is heated to about 50° C. Inthe process of an esterification catalyst. Water formed duringesterification is removed from the reaction mixture by distillation. Theprogress of the esterification reaction is monitored by measuring theconcentration of acrylic acid and/or methanol in the mixture.

Reactive with other monomers and imparting strength and durability toacrylic co-polymers, methyl acrylate is a useful monomer for coatingsfor leather, paper, floor coverings and textiles. Resins containingmethyl acrylate can be formulated as elastomers, adhesives, thickeners,amphoteric surfactants, fibers and plastics. Methyl Acrylate is alsoused in production of monomers used to make water treatment materialsand in chemical synthesis.

Example 28 Prophetic Example of Conversion of Acrylic Acid to EthylAcrylate

Acrylic acid, such as that provided in Example 19, is converted to ethylacrylate by direct, catalyzed esterification. Acrylic acid is contactedwith ethanol, and the mixture is heated to about 75° C. In the presenceof an esterification catalyst. Water formed during esterification isremoved from the reaction mixture by distillation. The progress of theesterification reaction is monitored by measuring the concentration ofacrylic acid and/or ethanol in the mixture.

Ethyl acrylate is used in the production of homopolymers and co-polymersfor use in textiles, adhesives and sealants. Ethyl acrylate is also usedin the production of co-polymers, for example acrylic acid and itssalts, esters, amides, methacrylates, acrylonitrile, maleates, vinylacetate, vinyl chloride, vinylidene chloride, styrene, butadiene andunsaturated polyesters. In addition, ethyl acrylate is used in chemicalsynthesis.

Example 29 Prophetic Example of Conversion of Acrylic Acid to ButylAcrylate

Acrylic acid, such as that provided in Example 22, is converted to butylacrylate by direct, catalyzed esterification. Acrylic acid is contactedwith 1-butanol, and the mixture is heated to about 100° C. In thepresence of an esterification catalyst. Water formed duringesterification is removed from the reaction mixture by distillation. Theprogress of the esterification reaction is monitored by measuring theconcentration of acrylic acid and/or ethanol in the mixture.

Butyl acrylate is used in the production of homopolymers and co-polymersfor use in water-based industrial and architectural paints, enamels,adhesives, caulks and sealants, and textile finishes, utilizinghomopolymers and co-polymers with methacrylates, acrylonitrile,maleates, vinyl acetate, vinyl chloride, vinylidene chloride, styrene,butadiene or unsaturated polyesters.

Example 30 Prophetic Example of Conversion of Acrylic Acid to EthylhexylAcrylate

Acrylic acid, such as that provided in Example 22, is converted toethylhexyl acrylate by direct, catalyzed esterification. Acrylic acid iscontacted with 2-ethyl-1 -hexanol, and the mixture is heated to about120° C. In the presence of an esterification catalyst. Water formedduring esterification is removed from the reaction mixture bydistillation. The progress of the esterification reaction is monitoredby measuring the concentration of acrylic acid and/or ethanol in themixture.

Ethylhexyl acrylate is used in the production of homopolymers andco-polymers for caulks, coatings and pressure sensitive adhesives,paints, leather finishing, and textile and paper coatings.

Example 31 Prophetic Example of Conversion of Acrylates to End Products,including Consumer Products

One or more acrylates as provided in Examples 24-27 is further convertedto one or more of adhesives, surface coatings, water-based coatings,paints, inks, leather finishes, paper coatings, film coatings,plasticizers, or precursors for flocculants. Such conversions to endproducts employ methods known in the art.

Example 32 Prophetic Example of Acrylic-based Paint Manufacture

An aqueous dispersion comprising at least one particulatewater-insoluble copolymer that includes one or more of acrylic acid,ethyl acrylate, methyl acrylate, 2-ethylhexyl acrylate, butyl acrylate,lauryl acrylate or other copolymer obtained from acrylic acid convertedfrom 3-HP microbially produced, as described elsewhere herein, isobtained by mixing such components together under sufficient agitationto form a stable dispersion of the copolymers. The copolymers have anaverage molecular weight that is at least 50,000, with the copolymerparticles having diameters in the range of 0.5 to 3.0 microns. Othercomponents in the aqueous dispersion may include pigment, filler (e.g.,calcium carbonate, aluminum silicate), solvent (e.g., acetone, benzol,alcohols, etc., although these are not found in certain no VOC paints),thickener, and additional additives depending on the conditions,applications, intended surfaces, etc.

In variations of such acrylic-based paints, co-polymers in addition tothe acrylic-based polymers may be added. Such other co-polymers mayinclude, but are not limited to vinyl acetate, vinyl fluoride,vinylidene chiloride, methacrylic acid, itaconic acid, maleic acid, andstyrene.

Example 33 Prophetic Example of Conversion of 3-HP to 1,3-Propanediol

Acrylic acid, such as that provided in Example 22, is converted to1,3-propanediol. 3-HP is hydrogenated in the presence of an unsupportedruthenium catalyst, in a liquid phase, to prepare 1,3-propartediol. Theliquid phase includes water and cyclohexane. The hydrogenation iscarried out continuously in a stirred tank reactor at a temperature ofabout 150° C. and a pressure of about 1000 psi. The progress ofhydrogenation is monitored by measuring the concentration of 3-HP and/orhydrogen in the reactor.

Example 34 Prophetic Example of Conversion of 3-HP to Malonic Acid

Acrylic acid, such as that provided in Example 22, is converted tomalonic acid by catalytic oxidation of 3-HP by a supported catalystcomprising Rh. The catalytic oxidation is carried out in a fixed-bedreactor operated in a trickle-bed procedure. In the trickle-bedprocedure the aqueous phase comprising the 3-HP starting material, aswell as the oxidation products of the same and means for the adjustmentof pH, and oxygen or an oxygen-containing gas can be conducted incounterflow. In order to achieve a sufficiently short reaction time, theconversion is carried out at a pH of about 8. The oxidation is carriedout at a temperature of about 40° C. Malonic acid is obtained in nearlyquantitative yields.

Example 35 Increased Copy of Genetic Elements in the 3HPTGC ConferTolerance to 3-HP

Data from a SCALES evaluation of library clone fitness related to 3-HPexposure, using the SCALES technique, affords clear evidence of therelevance as to 3-HP tolerance of a number of genes and enzymes. Fromthis data, and in view of fitness data from other portions of the3HPTGC, a broad view may be obtained that appropriate modifications ofany of the genes or enzymes of the 3-HPTGC and/or provision of nucleicacid sequences that provide an enzyme activity of such enzymes (withoutnecessarily encoding the entire enzyme) may result in an alteredenzymatic activity that leads to increased 3-HP tolerance.

The method used to measure 3-HP tolerance conferred by genes in the3HPTGC is summarized as follows.

Bacteria, Plasmids, and Library Construction

Wild-type Escherichia coli K12 (ATCC #29425) was used for thepreparation of genomic DNA. Six samples of purified genomic DNA weredigested with two blunt cutters Alul and Rsal (Invitrogen, Carlsbad,Calif. USA) for different respective times—10, 20, 30, 40, 50 and 60minutes at 37 C, and then were heat inactivated at 70 C for 15 minutes.Restriction digestions were mixed and the fragmented DNA was separatedbased on size using agarose gel electrophoresis. Respective DNAfragments of 0.5, 1, 2, 4 and greater than 8 kb sizes were excised fromthe gel and purified with a gel extraction kit (Qiagen) according tomanufacturer's instructions. Genomic libraries were constructed byligation of the respective purified fragmented DNA with the pSMART-LCKANvector (Lucigen, Middleton, Wis. USA) according to manufacturer'sinstructions. Each ligation product was then electroporated into E.Cloni 10G Supreme Electrocompetent Cells (Lucigen) and plated onLB+kanamycin. Colonies were harvested and plasmid DNA was extractedusing Qiagen HiSpeed Plasmid Midi Kit according to manufacturer'sinstructions. Purified plasmid DNA of each library was introduced intoEscherichia coli strain Mach1-T1® (Invitrogen, Carlsbad, Calif. USA) byelectroporation. These cultures, representing each library—0.5, 1,0,2.0, 4.0 and >8.0 kb of genomic DNA, were combined and incubated at 37 Cto a desired density, to an OD₆₀₀ of approximately 0.50. This combinedlibrary culture mixture was used for selection. (See section herein andalso see Lynch, M., Watencke, T E, Gill, R T, SCALES: multiscaleanalysis of library enrichment. Nature Methods, 2007. 4(87-93);Warnecke, T. E., Lynch, M. D., Karimpour-Fard, A., Sandoval, N., Gill,R. T., A genomics approach to improve the analysis and design or strainselections. Metabolic Engineering, 2008 10(154-156)). Mach1-T1®containing pSMART-LCKAN empty vector were used for all control studies.Growth curves were done in MOPS Minimal Medium (See Neidhardt, F.,Culture medium for enterobacteria. J Bacteriol, 1974. 119: p. 736-747).Antibiotic concentration was 20 ug kanamycin/mL.

3-HP Preparation

3-HP was obtained from TCI America (Portland, Oreg.). Significantacrylic acid and 2-oxydipropionic contamination was observed via HPLCanalysis. Samples were subsequently treated by diethyl ether extractionto remove acrylic acid and a portion of the 2-oxydipropioniccontaminants. Samples were then neutralized with 10 M NaOH to a final pHof 7.0. Considerable insoluble matter was observed at neutral pH atconcentrations in excess of approximately 35 g/L. Neutralized sampleswere centrifuged at 4000 rpm for 30 minutes at 4° C. The soluble 3-HPfraction was isolated from the thus-centrifuged insoluble matter andfurther analyzed by HPLC for a final quantification of concentration andpurity of the working stock solution. The working stock solution wasused for the selection and MIC evaluations in this example.

Selections

As noted herein, five representative genomic libraries were created fromE. coli K12 genomic DNA with defined insert sizes of 0.5, 1, 2, 4, and 8kb, each library was transformed into MACH1-T1® E. coli, cultured andthen mixed. The mixture was aliquoted into two 15 mL screw cap tubeswith a final concentration of 20 g/L 3-HP (TCI America) neutralized topH 7 with 10 M NaOH. The cell density of the selection cultures wasmonitored as they approached a final OD₆₀₀ of 0.3-0.4. The originalselection cultures were subsequently used to inoculate another round of15 mL MOPS minimal media+kanamycin+3-HP as part of a repeated batchselection strategy. Overall, a selection was carried out over 8 serialtransfer batches with a decreasing gradient of 3-HP over 60 hours. Moreparticularly, the 3-HP concentrations were 20 g 3-HP/L for serialbatches 1 and 2, 15 g 3-HP/L for serial batches 3 and 4, 10 g 3-HP/L forserial batches 5 and 6, and 5 g 3-HP/L for serial batches 7 and 8. Forserial batches 7 and 8 the culture media was replaced as the cultureapproached stationary phase to avoid nutrient limitations. (Also seeWarnecke, T. E., Lynch, M. D., Karimpour-Fard, A., Sandoval, N., Gill,R. T. , A genomics approach to improve the analysis and design of strainselections. Metabolic Engineering, 2008 10(154-156), incorporated byreference herein). Batch transfer times were adjusted as needed to avoida nutrient limited selection environment. Samples were taken at theculmination of each batch. Repeated batch cultures containing 3-HP weremonitored and inoculated over a 60 hour period to enhance theconcentration of clones exhibiting increased growth in the presence of3-HP. Samples were taken by plating 1 mL of the selected population ontoselective plates (LB with kanamycin) with each batch. Plasmid DNA wasextracted from each sample and hybridized to Affymetrix E. ColiAntisense GeneChip® arrays (Affymetrix, Santa Clara, Calif.) accordingto previous work (See Lynch, M., Warencke, T E, Gill, R T, SCALEs;multiscale analysis of library enrichment. Nature Methods, 2007,4(87-93)) and manufacturer's instructions.

Data Analysis

Data analysis was completed by utilizing SCALEs-appropriate softwaredescribed herein and also in Lynch, M., Warencke, T E, Gill, R. T,SCALEs: multiscale analysis of library enrichment. Nature Methods, 2007.4(87-93)). Fitness contributions from specific genomic elements werecalculated from the enrichment of each region as a fraction of theselected population, as was previously described (Lynch, M., Warencke, TE, Gill, R T, SCALES: multiscale analysis of library enrichment. NatureMethods, 2007. 4(87-93)). Briefly, plasmid DNA from samples taken at theculmination of each batch in the selection were hybridized to AffymetrixE. Coli Antisense GeneChip® arrays per above and data obtained from thiswas further analyzed. For each array, signal values corresponding toindividual probe sets were extracted from the Affymetrix data file andpartitioned into probe sets based on similar affinity values (Naef, F.and Magnasco, M. O., 2003, Solving the riddle of the bright mismatches:labeling and effective binding in oligonucelotide arrays. Phys. Rev. E68, 011906). Background signal for each probe was subtracted accordingto conventional Affymetrix algorithms (MAS 5.0). Non-specific noise wasdetermined as the intercept of the robust regression of the differenceof the perfect match and mismatch signal against the perfect matchsignal. Probe signals were then mapped to genomic position as the tukeybi-weight of the nearest 25 probe signals and were de-noised by applyinga medium filter with a 1000 bp window length. Gaps between probes werefilled in by linear interpolation. This continuous signal was decomposedusing an N-sieve based analysis and reconstructed on a minimum scale of500 bp as described in detail by Lynch et al (2007). Signals werefurther normalized by the total repressor of primer (ROP) signal, whichis on the library vector backbone and represents the signalcorresponding to the total plasmid concentration added to the chip.

The analysis decomposed the microarray signals into correspondinglibrary clones and calculated relative enrichment of specific regionsover time. In this way, genome-wide fitness (ln(X_(i)/X_(i0))) wasmeasured based on region specific enrichment patterns for the selectionin the presence of 3-HP. Genetic elements and their correspondingfitness were then segregated by metabolic pathway based on their EcoCycclassifications (ecocyc.org). This fitness matrix was used to calculateboth pathway fitness (W) and frequency of enrichment found in theselected population.

$W_{pathway} = {\sum\limits_{1}^{n}W_{i}}$${frequency} = \frac{{number}\mspace{14mu} {of}\mspace{14mu} {genes}\mspace{14mu} {from}\mspace{14mu} {metabolic}\mspace{14mu} {pathway}}{{total}\mspace{14mu} {genes}\mspace{14mu} {in}\mspace{14mu} {pathway}}$

Pathway redundancies were identified by an initial rank ordering ofpathway fitness, followed by a specific assignment for genetic elementsassociated with multiple pathways to the primary pathway identified inthe first rank, and subsequent removal of the gene-specific fitnessvalues from the secondary pathways.

Similarly genes in a given genetic element were assigned fitnessindependent of neighboring genes in a genetic element as follows: Thefitness of any gene was calculated as the sum of the fitness of allclones that contained that gene. This was followed by an initial rankordering of gene fitness, followed by a specific assignment for geneticelements associated with multiple genes to the dominant gene identifiedin genetic element with the highest rank, with the subsequent removal ofthe fitness values from the non dominant genes in a genetic element.

Data was further analyzed by construction of receiver operatorcharacteristics (“ROC”) according to traditional signal detection theory(T. Fawcett, “An introduction to ROC analysis,” Pattern Recog. Let.(2006)27:861-874). Data was categorized according to four standardclasses—true positive, false positive, true negative, and falsenegative, using the fitness values for respective genetic elements perabove and specific growth rates measured in the presence of 20 g/L 3-HP,using standard methods of analysis and cutoff values for fitness of 0.1,1.0, 10 and 20 were chosen in an effort to optimize the range of trueand false positive rates. A data point representing a genetic element ofa clone was denoted a true positive if the reported fitness was greaterthan the cutoff value and the separately measured growth rate wassignificantly increased when compared with the negative control. A falsepositive had reported fitness that was greater than the cutoff value buta growth rate not significantly greater than that of the negativecontrol. A clone was designated a true negative only if thecorresponding fitness was less than the cutoff value and it yieldedsignificantly reduced growth rates, i.e., not significantly greater thanthat of the negative control, and a false negative refers to a clonehaving a reduced fitness score but demonstrating an increased growthrate, i.e., significantly greater than that of the negative control.

An ROC curve is constructed by plotting the true positive rate(sensitivity) versus the false positive rate (1-specificity) (See T. E.Warnecke et al. Met. Engineering 10 (2008):154-165). Accordingly, it maybe stated with confidence that clones (and their respective geneticelements) identified with increased fitness confer tolerance to 3-HPover the control.

Results

FIGS. 9B-9G, graphically show the genes identified in the 3HPTGC for E.coli. In addition Table 3 gives cumulative fitness values as calculatedherein for some of the genes in the 3HPTGC.

3-HP Toleragenic Complexes also were developed for the gram-positivebacterium Bacillus subtilis, for the yeast Saccharomyces cerevisiae, andfor the bacterium Cupriavidus necator. These are depicted, respectively,in FIGS 9B-D, sheets 1-7.

Example 36 Additions of 3HPTGC Products, Part 1

Based on the examples, and conceptualization of the 3HPTGC, it ispossible to increase the 3-HP tolerance of a microorganism by addinglimiting enzymatic conversion products product(s) of an enzymaticconversion step) of the 3HPTGC. This example demonstrates the additionof some such products to increase 3-HP tolerance in E. coli.

Bacteria, Plasmids, and Media

Wild-type Escherichia coli K12 (ATCC #29425) was used for thepreparation of genomic DNA. Mach1-T1® was obtained from Invitrogen(Carlsbad, Calif. USA).

3-HP Preparation

3-HP was obtained front TC1 America (Portland, Oreg.). Significantcrylic acid and 2-oxydipropionic contamination was observed via HPLCanalysis, Samples were subsequently treated by diethyl ether extractionto remove acrylic acid and a portion of the 2-oxydipropioniccontaminants. Samples were then neutralized with 10 M NaOH to a final pHof 7.0. Considerable 3-HP polymerization was observed at neutral pH atconcentrations in excess of approximately 35 g/L. Neutralized sampleswere centrifuged at 4000 rpm for 30 minutes at 4° C. The soluble 3-HPfraction was isolated from the solid polymer product and furtheranalyzed by HPLC for a final quantification of concentration and purityof the working stock solution. The working stock solution was used forthe selection, growth rates and MIC evaluations in this example.

Minimum Inhibitory Concentrations

The minimum inhibitory concentration (MIC) using commercially obtained3-HP (TC1 America, Portland, Oreg. USA, see 3-HP preparation herein) wasdetermined microaerobically in a 96 well-plate format. Overnightcultures of strains were grown in 5 ml LB (with antibiotic whereappropriate). A 1 v/v % was used to inoculate a 15 ml conical tubefilled to the top with MOPS minimal media and capped. After the cellsreached mid exponential phase, the culture was diluted to an OD₆₀₀ of0.200. The cells were further diluted 1:20 and a 10 ul aliquot was usedto inoculate each well (˜10⁴ cells per well). The plate was arranged tomeasure the growth of variable strains or growth conditions inincreasing 3-HP concentrations, 0-70 g/L, in 5 g/L increments, as wellas either media supplemented with optimal supplement concentrationswhich were determined to be: 2.4 mM tyrosine (Sigma), 3.3 mMphenylalanine (Sigma), 1 mM tryptophan (Sigma), 0.2 mM p-hydroxybenzoicacid hydrazide (MP Biomedicals), 0.2 mM p-aminobenzoic acid (MPBiomedicals), 0.2 mM, 2,3-dihydroxybenzoic acid (MP Biomedicals), 0.4 mMshikimic acid (Sigma), 2 mM pyridoxine hydrochloride (Sigma), 35 uMhomoserine (Acros), 45 uM homocysteine thiolactone hydrochloride (MPBiomedicals), 0.5 mM oxobutanoate (Fluka), 5 mM threonine (Sigma). Theminimum inhibitory 3-HP concentration (i.e., the lowest concentration atwhich there is no visible growth) and the maximum 3-HP concentrationcorresponding to visible cell growth (OD˜0.1) were recorded after 24hours (between 24 and 25 hours, although data indicated no substantialchange in results when the le period was extended).

Results

3-HP tolerance of E. coli Mach1-T1® was increased by adding thesupplements to the media. The supplementation described herein resultedin the following MIC increases: 40% (tyrosine), 33% (phenylalanine), 33%(tryptophan), 33% (p-hydroxybenzoic acid hydrazide), 7% (p-aminobenzoicacid), 33% (2,3-didyroxybenzoic acid), 0% (pyridoxine hydrochloride),33% (homoserine), 60% (homocysteine thiolactone hydrochloride), 7%(oxobutanoate), and 3% (threonine).

Example 37 Additions of 3HPTGC Products, Part 2 (Using New Source of3-HP)

Based on the examples, and conceptualization of the 3HPTGC, it ispossible to increase the 3-HP tolerance of a microorganism by addinglimiting enzymatic conversion products (at least some of whichalternatively may be termed “intermediates”) of the 3HPTGC. This exampledemonstrates the addition of putrescine, spermidine, cadaverine andsodium bicarbonate to increase 3-HP tolerance in E. coli. The concept of‘limiting’ as used in this context refers to a hypothesized limitationthat if overcome may demonstrate increased 3-HP tolerance by a subjectmicroorganism or system. As a non-exclusive approach, such hypothesizedlimitation may be confirmed experimentally, as by a demonstration ofincreased tolerance to 3-HP upon addition of a particular enzymaticconversion product or other compound.

Bacteria, Plasmids, and Media

Wild-type Escherichia coli K12 (ATCC #29425) was used for thepreparation of genomic DNA. M9 minimal and EZ rich media are describedin Subsection II of the Common Methods Section.

3-HP Preparation

3-HP was obtained from Beta-propiolactone as described in Subsection IIIof the Common Method Section.

Minimum Inhibitory Concentrations

The minimum inhibitory concentration (MIC) of 3-HP for E. coli (see 3-HPpreparation herein) was determined aerobically in a 96 well-plateformat. Overnight cultures of strains were grown in 5 ml LB (withantibiotic where appropriate) at 37° C. In a shaking incubator. A 1 v/v% was used to inoculate 10 mL of M9 minimal media. After the cellsreached mid exponential phase, the culture was diluted to an OD₆₀₀ of0.200. The cells were further diluted 1:20 and a 10 ul aliquot was usedto inoculate each well (˜10⁴ cells per well). The plate was arranged tomeasure the growth of variable strains or growth conditions inincreasing 3-HP concentrations, 0-100 g/L, in 10 g/L increments, in M9minimal media, supplemented with putrescine (0.1 g/L, MP Biomedicals,Santa Ana, Calif. USA), cadaverine (0.1 g/L, MP Biomedicals) orspermidine (0.1 g/L, Sigma-Aldrich, St. Louis, Mo., USA) or sodiumbicarbonate (20 mM, Fisher Scientific, Pittsburgh, Pa. USA) (values inparentheses indicate final concentrations in media). The minimuminhibitory 3-HP concentration (i.e., the lowest concentration at whichthere is no visible growth) and the maximum 3-HP concentrationcorresponding to visible cell growth (OD˜0.1) were recorded after 24hours (between 24 and 25 hours, although data (not shown) indicated nosubstantial change in results when the time period was extended). TheMIC endpoint is the lowest concentration of compound at which there wasno visible growth.

Results

3-HP tolerance of E. coli was increased by adding the polyaminesputrescine, spermidine and cadaverine to the media. Minimum inhibitoryconcentrations (MICS) for E. coli K12 in control and supplemented mediawere as follows: in M9 minimal media supplemented with putrescine 40g/L, in M9 minimal media supplemented with spermidine 40 g/L, in M9minimal media supplemented with cadavarine 30 g/L. Minimum inhibitoryconcentrations (MICs) for added sodium bicarbonate in M9 minimal mediawas 30 g/L. The Minimum inhibitory concentrations (MICS) for E. coli K12in 100 g/L stock solution 3-HP was 20 g/L.

In view of the increase over the control MIC with sodium bicarbonatesupplementation, other alteration, such as regulation and/or geneticmodification of carbonic anhydrase (not presently shown in FIGS. 9B-G,but related directly to HCO₃ ⁻), such as providing a heterologousnucleic acid sequence to a cell of interest, where that nucleic acidsequence encodes a polypeptide possessing carbonic anhydrase activityare considered of value to increase tolerance to 3-HP (such as incombination with other alterations of the 3HPTGC). Similarly, and assupported by other data provided herein, alterations of the enzymaticactivities, such as by genetic modification(s) of enzyme(s) along the3HPTGC pathway portions that lead to arginine, putrescine, cadaverineand spermidine, are considered of value to increase tolerance to 3-HP(such as in combination with other alterations of the 3HPTGC).

Example 38 Genetic Modification of aroH for Increased 3-HP Tolerance

Based on the identification of the tyrA-aroF operon as a genetic elementconferring tolerance to 3-HP at increased copy, this enzymatic activitywas further examined. The wild type aroF gene is inhibited by increasingconcentrations of end products tyrosine and phenylalanine. However, tobypass this inherent feedback inhibition control, a feedback resistantmutant of the aroH gene was obtained and introduced into a cell asfollows.

Clone Construction

PCR was used to amplify the E. coli K12 genomic DNA corresponding to thearoF-tyrA region with primers designed to include the upstream aroFppromoter and the rho-independent transcriptional terminators. Ligationof the purified, fragmented DNA with the pSMART-kanamycin vectors wasperformed with the CloneSMART kit (Lucigen, Middleton, Wis. USA)according to manufacturer's instructions. The ligation product was thentransformed into chemically competent Mach1-T1® E. coli cells(Invitrogen, Carlsbad, Calif. USA), plated on LB+kanamycin, andincubated at 37° C. for 24 hours. To confirm the insertion of positivetransformants, plasmids were isolated from clones using a Qiaprep SpinMiniPrep Kit from Qiagen (Valencia, Calif.) and sequenced (Macrogen,South Korea).

Plasmids containing the wild-type aroH gene (CB202) and a mutant versionexhibiting resistance to tryptophan feedback inhibition (CB447) via asingle amino acid change (G149D) were obtained from Ray et al (Ray, J.M., C. Yanofsky, and R. Baurele, Mutational analysis of the catalyticand feedback sites of the tryptophan-sensitive3-deoxy-D-arabino-heptulosante-7-phosphate synthase of Escherichia coli.T Bacteriol, 1988. 170(12):p. 5500-6.). These plasmids were constructedwith the pKK223-3 backbone vector containing the ptac promoter andrrNBT1 transcriptional terminator. The aroH inset DNA was amplifiedaccording to traditional PCR methodology with primers designed toinclude both the promoter and terminator. Purified PCR products wereligated with the pBT-1 plasmid and transformed into electrocompetentMach1-T1® (Lynch, M. D. and R. T. Gill, A series of broad host rangevectors for stable genomic library construction. Biotechnology andBioengineering, 2006. 94(1): p. 151-158). The resulting plasmid sequenceis given in (SEQ ID NO:001). Optimal induction levels were determined byminimum inhibitory concentration assays to be 0.001 mM IPTG.

MIC Comparisons

MIC evaluations were conducted as described for Example 35. A Mach1-T1®cell culture comprising the aroH mutant was compared with a control cellculture, both in MOPS minimal media.

Results

As measured by fold increase in MIC, the cells comprising the aroHmutant exhibited a MIC 1.4 times greater than the control MIC. Thisrepresents a 40 percent improvement. Accordingly, this exampledemonstrates one of many possible genetic modification approaches toincreasing 3-HP tolerance in a selected cell, based on knowledge of theimportance of the 3HPTGC in 3-HP tolerance.

Example 39 Genetic Modification via Cyanase Introduction for Increased3-HP Tolerance

A plasmid clone containing the cynTS genes from E. coli K12 was obtainedfrom selections described in Example 35. This plasmid calledpSMART-LC-Kan-cynTS was isolated and purified according to standardmethods. (Sequencing of the plasmid revealed a final sequence (SEQ IDNO:002)). Purified plasmid was retransformed into E. coli K12 bystandard techniques and MIC measured as described in Example 37. 3-HPtolerance improvement by the plasmid containing the cynTS genes.

Minimum inhibitory concentrations (MICS) of 3-HP for E. coli K12 and E.coli K12+pSMART-LC-Kan-cynTS in M9 minimal media were 30 g/L, and 50g/L, respectively. Thus, an over sixty percent improvement in the MIC,signifying an increase in 3-HP tolerance, was observed in this examplewhich comprised only one genetic modification of the 3HPTGC in the E.coli host cell. Accordingly, this example again demonstrates one of manypossible genetic modification approaches to increasing 3-HP tolerance ina selected cell, based on knowledge of the importance of the 3HPTGC in3-HP tolerance and appropriate use of that knowledge.

Example 40 Development of a Nucleic Acid Sequence Encoding a ProteinSequence Comprising Oxaloacetate Alpha—Decarboxylase Activity (PartialProphetic)

Several 2-keto acid decarboxylases with a broad substrate range havebeen previously characterized (Pohl, M., Sprenger, G. A., Muller, M., Anew perspective on thiamine catalysis. Current Opinion in Biotechnology,15(4), 335-342 (2004)). Of particular interest is an enzyme from M.tuberculosis, alpha-ketoglutarate decarboxylase, which has been purifiedand characterized (Tian, J., Bryk, R. Itoh, M., Suematsu, M., and CarlNathan, C. Variant tricarboxylic acid cycle in Mycobacteriumtuberculosis: Identification of alpha-ketoglutarate decarboxylase. PNAS.Jul. 26, 2005 vol. 102(30): 10670-10677;; Stephanopoulos, G., Challengesin engineering microbes for biofuels production. Science, 2007.315(5813):801-804). The reaction carried out by this enzyme is depictedin FIG. 16B (FIG. 16A showing the predominant known chemical reaction bythe enzyme encoded by the native kgd gene). The native kgd gene haspreviously been cloned, expressed and purified from E. coli withouttechnical difficulty or toxic effects to the host strain (Tian, J.,Bryk, R. Itoh, M., Suematsu, M., and Carl Nathan, C. Varianttricarboxylic acid cycle in Mycobacterium tuberculosis: Identificationof alpha-ketoglutarate decarboxylase. PNAS. Jul. 26, 2005 vol.102(30):10670-10677; Stephanopoulos, G., Challenges in engineeringmicrobes for biofuels production. Science, 2007. 315(5813):801-804).This enzyme has also been chosen as it is unlikely to be associated withthe alpha-ketoglutarate dehydrogenase. Of additional interest is that aconvenient colorimetric method has been developed to assay thisenzymatic activity. The kgd enzyme is evolved as provided herein to havea measurable, enzymatic function depicted in FIG. 16B, thedecarboxylation of oxaloacetate to malonate semialdehyde. The technicalwork to achieve this relies largely upon traditional selection andscreening of mutants of the alpha-keto-glutarate decarboxylase that havethe desired oxaloacetate alpha-decarboxylase activity.

As a first step a mutant library is constructed of the kgd gene thatwill be used for selections or screening. The protein sequence for thealpha-ketoglutarate decarboxylase from M. tuberculosis was codonoptimized for E. coli according to a service from DNA 2.0 (Menlo Park,Calif. USA), a commercial DNA gene synthesis provider. The nucleic acidsequence was synthesized with an eight amino acid N-terminal tag toenable affinity based protein purification. This gene sequenceincorporated an NcoI restriction site overlapping the gene start codonand was followed by a HindIII restriction site. In addition a ShineDelgarno sequence (i.e., a ribosomal binding site) was placed in frontof the start codon preceded by an EcoRI restriction site. This geneconstruct was synthesized by DNA 2.0 and provided in a pJ206 vectorbackbone.

A circular plasmid based cloning vector termed pKK223-kgd for expressionof the alpha-ketoglutarate decarboxylase in E. coli was constructed asfollows. Plasmid DNA pJ206 containing the gene synthesized kgd gene wassubjected to enzymatic restriction digestion with the enzymes EcoRI andHindIII obtained from New England BioLabs (Ipswich, Mass. USA) accordingto manufacturer's instructions. The digestion mixture was separated byagarose gel electrophoresis, and visualized under UV transilluminationas described in Subsection II of the Common Methods Section. An agarosegel slice containing a DNA piece corresponding to the kgd gene was cutfrom the gel and the DNA recovered with a standard gel extractionprotocol and components from Qiagen according to manufacturer'sinstructions. An E. coli cloning strain bearing pKK223-aroH was obtainedas a kind a gift from the laboratory of Prof. Ryan T. Gill from theUniversity of Colorado at Boulder. Cultures of this strain bearing theplasmid were grown by standard methodologies and plasmid DNA wasprepared by a commercial miniprep column from Qiagen (Valencia, Calif.USA) according to manufacturer's instructions. Plasmid DNA was digestedwith the restriction endonucleases EcoRI and HindIII obtained from NewEngland BioLabs (Ipswich, Mass. USA) according to manufacturer'sinstructions. This digestion served to separate the aroH reading framefrom the pKK223 backbone. The digestion mixture was separated by agarosegel electrophoresis, and visualized under UV transillumination asdescribed in Subsection II of the Common Methods Section. An agarose gelslice containing a DNA piece corresponding to the backbone of the pKK223plasmid was cut from the gel and the DNA recovered with a standard gelextraction protocol and components from Qiagen (Valencia, Calif. USA)according to manufacturer's instructions.

Pieces of purified DNA corresponding to the kgd gene and pKK223 vectorbackbone were ligated and the ligation product was transformed viaelectroporation according to manufacturer's instructions. The sequenceof the resulting vector termed pKK223-kgd (SEQ ID NO:004) was confirmedby routine sequencing performed by the commercial service provided byMacrogen (Rockville, Md. TJSA). pKK223-kgd confers resistance tobeta-lactamase and contains the kgd gene of M. tuberculosis undercontrol of a ptac promoter inducible in E. coli hosts by IPTG.

Plasmid pKK223-kgd was propagated and purified DNA prepared by standardmethodologies. Plasmids were introduced into XL1-Red chemicallycompetent cells (Stratagene, LaJolla, Calif.) In accordance with themanufacturer's instructions, plated onto LB+100 micrograms/mLampicillin, and incubated at 37° C. for >24 hours. Dilution cultureswith 1/1000 of the original transformation volume were plated on LB+100micrograms/mL ampicillin in triplicate. Greater than 1000 colonies wereobtained, corresponding to approximately 10⁷ mutant cells pertransformation. Colonies were harvested by gently scraping the platesinto TB media. The cultures were immediately resuspended by vortexing,and aliquoted into 1 mL freezer stock cultures with a final glycerolconcentration of 15% (v/v) (Sambrook and Russell, 2001). The remainderof the culture was pelleted by centrifugation for 15 minutes at 3000rpm. Plasmid DNA was extracted according to the manufacturer'sinstructions using a HiSpeed Plasmid Midi Kit (Qiagen, Valencia,Calif.). Purified plasmid DNA from each mutant library was introducedinto E. coli 10GF′ (Lucigen, Middleton, Wis. USA) by electroporation.1/1000 volume of this transformation was plated on LB+kanamycin intriplicate to determine transformation efficiency and adequatetransformant numbers (>10̂6).

The selection based approach described herein allows for the rapididentification of a kgd mutant with oxaloacetate alpha-decarboxylaseactivity. An available strain of E. coli, strain AB354, is used as ahost for the selection (Bunch, P. K., F. Mat-Jan, N. Lee, and D. P.Clark. 1997. The ldhA gene encoding the fermentative lactatedehydrogenase of Escherichia coli. Microbiology 143:187-195). Thisauxotrophic E. coli strain has a mutation in panD, encoding aspartatedecarboxylase. The product of this reaction, beta-alanine is anessential intermediate in the synthesis of pantothenate, a precursor tocoenzyme A. The block in coenzyme A synthesis confers an inability ofthis E. coli strain to grow on minimal media without supplementation(Cronoan, J. E., Little, K. J., Jackowski, S.; Genetic and BiochemicalAnalyses of Pantothenate Biosynthesis in Escherichia coli and Salmonellatyphimurium, J. of Bacteriology, 149(3), 916-922 (1982); Cronan, J. E.,Beta-Alanine Synthesis in Escherichia coli J. of Bacteriology, 141(3),1291-1297 (1980)). The expression of gabT from R. norvegicus confersbeta-alanine aminotransferase activity to E. coli (Tunnicliff, G.; Ngo,T. T.; Rojo-Ortega, J. M.; Barbeau, A.; The inhibition by substrateanalogues of gamma-aminobutyrate aminotransferase from mitochondria ofdifferent subcellular fractions of rat brain Can. J. Biochem. 55,479-484 (1977)). This enzyme can utilize malonate semialdehyde as asubstrate to produce beta-alanine. A strain of E. coli AB354 expressinggabT (E. coli AB354+gabT) in addition to a mutant kgd gene havingoxaloacetate alpha-decarboxylase activity is capable of producing themetabolite beta-alanine and have a restored ability to grown on minimalmedia. Expected results of the selection are depicted in FIG. 18.

Similar to the kgd gene, a codon and expression optimized R. norvrgicusgabT gene is obtained via gene synthesis from the commercial providerDNA 2.0 (Menlo Park, Calif. USA), It is subsequently cloned into anexpression plasmid.

The mutant library of kgd genes is introduced into E. coli strain AB354expressing the gabT gene. This population will then be grown on minimalmedia plates. Individual mutants expressing the desired oxaloacetatealpha-decarboxylase activity are expected to show a restored ability toform colonies under these conditions. These clones are isolated and themutant proteins they express subsequently are selected for oxaloacetatealpha-decarboxylase activity.

With the successful construction selection of a mutant kgd library foroxaloacetate alpha-decarboxylase activity, it will be necessary toconfirm that these mutants have the desired enzymatic activity. Thus,mutants positive for oxaloacetate alpha-decarboxylase activity areconfirmed for alpha-decarboxylase activity. To accomplish this, acolorimetric screening approach is taken from current standardmethodologies. This approach is illustrated in FIG. 19. This approachnecessitates the expression and purification of the mutant enzymes andreaction with the purified enzyme, its cofactor (thiamin pyrophosphate)and the appropriate substrate. Protein expression and purification isperformed with standard methodologies.

Example 41 One-Liter Scale Bio-Production of 3-HP using E. coliDF40+pKK223+MCR

Using E. coli strain DF40+pKK223+MCR that was produced in accordancewith Example 1, a hatch culture of approximately 1 liter working volumewas conducted to assess microbial bio-production of 3-HP.

E. coli DF40+pKK223+MCR was inoculated from freezer stocks by standardpractice (Sambrook and Russell, 2001) into a 50 mL baffled flask of LBmedia plus 200 μg/mL ampicillin where indicated and grown to stationaryphase overnight at 37° C. with shaking at 225 rpm. In the morning, thisculture was used to inoculate (5% v/v) a 1-L bioreactor vesselcomprising M9 minimal media plus 5% (w/v) glucose plus 200 μg/mLampicillin, plus 1 mM IPTG, where indicated. The bioreactor vessel wasmaintained at pH 6.75 by addition of 10 M NaOH or 1 M HCl, asappropriate. The dissolved oxygen content of the bioreactor vessel wasmaintained at 80% of saturation by continuous sparging of air at a rateof 5 L/min and by continuous adjustment of the agitation rate of thebioreactor vessel between 100 and 1000 rpm. These bio-productionevaluations were conducted in at least triplicate. To monitor growth ofthese cultures, optical density measurements (absorbance at 600 nm, 1 cmpath length), which correlates to cell number, were taken at the time ofinoculation and every 2 hrs after inoculation for the first 12 hours. Onday 2 of the bio-production event, samples for optical density and othermeasurements were collected every 3 hours. For each sample collected,cells were pelleted by centrifugation and the supernatant was collectedfor analysis of 3-HP production as described per “Analysis of culturesfor 3-HP production” in the Common Methods section. Preliminary finaltiter of 3-HP in this 1-liter bio-production volume was calculated basedon HPLC analysis to be 0.7 g/L 3-HP. It is acknowledged that there islikely co-production of malonate semialdehyde, or possibly anotheraldehyde, or possibly degradation products of malonate semialdehyde orother aldehydes, that are indistinguishable from 3-HP by this HPLCanalysis.

Example 42 Tolerance Plus Bio-Production Pathway (Prophetic Example)

Using methods known to those skilled in the art, including thoseprovided in the Common Methods Section, and also using specific methodsfrom the other examples herein as to making and incorporating nucleicacid sequences to provide increased 3-HP tolerance and to provide 3-HPbio-production, genetic modifications are made to a selectedmicroorganism to provide heterologous nucleic acid sequences thatincrease both 3-HP tolerance and 3-HP production above levels found inthe non-modified microorganism. A plasmid or other vector or a DNAsequence (for direct incorporation) is constructed that comprises one ormore nucleic acid sequences that encode for enzyme(s) or otherpolypeptide(s) that, when combined into and expressed in the selectedmicroorganism, increase(s) tolerance to 3-HP by modifying one or moreaspects of the 3HPTGC. That or a different plasmid or other vector or aDNA sequence (for direct incorporation) is constructed to comprise oneor more nucleic acid sequences that encode for enzymes) or otherpolypeptide(s) that, when expressed in the selected microorganism,provide for (or increase) 3-HP bio-production.

In the case of plasmids, the plasmid(s) is/are contacted with theselected microorganism under suitable conditions to promotetransformation, and transformed microorganisms are selected for andidentified. In the case of other vectors or the DNA sequence(s), theseare introduced to the selected microorganism by methods well-known tothose skilled in the art. Selection for transformed recombinantmicroorganisms likewise may be conducted according to methods well-knownto those skilled in the art.

A first particular resultant recombinant microorganism comprisesenhanced 3-HP tolerance and bio-production capabilities compared to thecontrol, non-tolerance-modified microorganism, in which 3-HP toleranceis at least 20 percent greater than tolerance of thenon-tolerance-modified control and 3-HP bio-production is at least 20percent greater than 3-HP bio-production of the non-tolerance-modifiedcontrol. 3-HP tolerance is assessed by a 24-hour Minimum InhibitoryConcentration (MIC) evaluation based on the MIC protocol provided in theCommon Methods Section. 3-HP bio-production is based on a batch culturecomparison lasting for at least 24 hours past lag phase, and final 3-HPtiters are determined using the HPLC methods provided in the CommonMethods Section.

Example 43 Demonstration of Suitable Metrics for Comparison of ToleranceImprovements

Growth rate data was determined for the following species under thespecified conditions, aerobic and anaerobic, across a range of 3-HPconcentrations in the cell cultures. This demonstrates methods that maybe used to assess differences between a control and a treatmentmicroorganism. These or other methods may be used to demonstratetolerance differences for various embodiments of the present invention.

As shown in the accompanying figures, FIGS. 15A-O, the data may beevaluated and presented in a number of ways: a “toleragram” (showinggrowth rates at different 3-HP concentrations); change in opticaldensity over the evaluation period; and number of cell doublings overthe evaluation period.

These are provided to indicate non-limiting methodologies and approachesto assessing changes in tolerance, including microorganism and culturesystem tolerance, in addition to the use of MIC evaluations.

The following methods were used to generate the data in the notedfigures.

E. coli Aerobic

Overnight cultures of wild-type E. coli BW25113 were gown in triplicatein 5 mL standard LB medium. 100 uL of overnight cultures were used toinoculate triplicate 5 mL samples of M9 minimal medium+3HP, containing47.7 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.6 mM NaCl, 18.7 mM NH₄Cl, 2 mM MgSO₄,0.1 mM CaCl₂, and 0.4% glucose, with 3HP concentrations ranging from0-50 g/L. Starting OD₆₀₀ ranged from 0.02-0.08. Cultures were incubatedat 37 C for about 24 hours, and OD₆₀₀ was recorded every 1-2 hours forthe first 8 hours with a final OD₆₀₀ recorded at about 24 hours. Maximumspecific, growth rates (μ_(max)) were calculated by determining theoptimal fit of exponential trend lines with OD data for the evaluationperiod. Specific changes in OD₆₀₀ over approximately 24 hours(Δ_(24 hr)OD₆₀₀) were calculated as the difference in t=24 hr and t=0optical density, Δ_(24 hr)OD₆₀₀=(OD_(t=24))−(OD_(t=0)). Specific numberof doublings (N_(d)) were calculated by solving for N in the equation2^(N)=(OD_(t=24))/(OD_(t=0)).

E. coli Anaerobic

Overnight cultures of wild-type E. coli BW25113 were gown in triplicatein 5 mL standard LB medium. 100 uL of overnight cultures were used toinoculate triplicate 5 mL samples of M9 minimal medium+3HP, containing47.7 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.6 mM NaCl, 18.7 mM NH₄Cl, 2 mM MgSO₄,0.1 mM CaCl₂, and 0.4% glucose, with 3HP concentrations ranging from0-50 g₁'L. Starting OD₆₀₀ ranged from 0.02-0.08. Cultures were spargedwith CO₂ for 10 seconds, sealed, and incubated at 37 C for about 24hours. OD₆₀₀ was recorded every 1-2 hours during the first 8 hours witha final OD₆₀₀ recorded at about 24 hours. For each data point the samplewas opened, sampled, re-sparged with CO₂, and sealed once again. Maximumspecific growth rates (μ_(max)) were calculated by determining theoptimal fit of exponential trend lines with OD data for the evaluationperiod. Specific changes in OD₆₀₀ over approximately 24 hours(Δ_(24 hr)OD₆₀₀) were calculated as the difference in t=24 hr and t=0optical density, Δ_(24 hr)OD₆₀₀=(OD_(t=24))−(OD_(t=0)). Specific numberof doublings (N_(d)) were calculated by solving for N in the equation2^(N)=(OD_(t=24))/(OD_(t=0)).

Bacillus Subtilis Aerobic

Overnight cultures of wild-type B. Subtilis were grown in triplicate in5 mL standard LB medium. 100 uL of overnight cultures were used toinoculate triplicate 5 mL samples of M9 minimal medium+3HP+glutamatesupplementation, containing 47.7 mM Na₂HPO, 22 mM KH₂PO₄, 8.6 mM NaCl,18.7 mM NH₄Cl, 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glucose, and 10 mMglutamate, with 3HP concentrations ranging from 0-50 g/L. Starting OD₆₀₀ranged from 0.02-0.08. Cultures were incubated at 37C for about 24hours, and OD₆₀₀ was recorded every 1-2 hours for the first 8 hours witha final OD₆₀₀ recorded at about 24 hours. Maximum specific growth rates(μ_(max)) were calculated by determining the optimal fit of exponentialtrend lines with OD data for the evaluation period. Specific changes inOD₆₀₀ over approximately 24 hours (Δ_(24 hr)OD₆₀₀) were calculated asthe difference in t=24 hr and t=0 optical density, Δ24 hrOD₆₀₀=(OD_(24 hr))−(OD_(t=0)). Specific number of doublings (N_(d)) werecalculated by solving for N in the equation2^(N)=(OD_(t=24))/(OD_(t=o)).

S. cerevisiae Aerobic

Overnight cultures of S. cerevisiae were grown in triplicate in 5 mLstandard YPD medium containing 10 g/L, yeast extract, 20 g/L peptone,and 2% glucose. 100 uL of overnight cultures were used to inoculatetriplicate 5 mL samples of SD minimal medium (without vitamins)+3HP,containing 37.8 mM (NH₄)₂SO₄, 8.1 uM H₃B₃, 0.25 uM CuSO₄, 0.6 uM Kl,1.25 uM FeCl₃, 2.65 uM MnSO₄, 1 uM Na₂MoO₄, 2.5 uM ZnSO₄, 6.25 mMKH₂PO₄, 0.86 mM K₂HPO₄, 4.15 mM MgSO₄, 1.71 mM NaCl, 0.90 mM CaCl₂, and2% glucose, with 3HP concentrations ranging from 0-50 g/L. StartingOD₆₀₀ ranged from 0.03-0.08. Cultures were sparged with CO₂ for 10seconds, sealed, and incubated at 30 C for about 24 hours. OD₆₀₀ wasrecorded every 1-2 hours for the first 8-12 hours with a final OD₆₀₀recorded at about 24 hours. Maximum specific growth rates (μ_(max)) werecalculated by determining the optimal fit of exponential trend lineswith OD data for the evaluation period. Specific changes in OD₆₀₀ overapproximately 24 hours (Δ_(24 hr)OD₆₀₀) were calculated as thedifference in t=24 hr and t=0 optical density,Δ_(24 hr)OD₆₀₀=(OD_(t=24))−(OD_(t=0)). Specific number of doublings(N_(d)) were calculated by solving for N in the equation2^(N)=(OD_(t=)24)/(OD_(t=0)).

S. cerevisiae Anaerobic

Overnight cultures of S. cerevisiae were grown in triplicate in 5 mLstandard YPD medium containing 10 g/L yeast extract, 20 g/L peptone, and2% glucose. 100 uL of overnight cultures were used to inoculatetriplicate 5 mL samples of SD minimal medium (without vitamins)+3HP,containing 37.8 mM (NH₄)₂SO₄, 8.1 uM H₃BO₃, 0.25 uM CuSO₄, 0.6 uM Kl,1.25 uM FeCl₃, 2.65 uM MnSO₄, 1 uM Na₂MoO₄, 2.5 uM ZnSO₄, 6.25 mMKH₂PO₄, 0.86 mM K₂HPO₄, 4.15 mM MgSO₄, 1.71 mM NaCl, 0.90 mM CaCl₂, and2% glucose, with 3HP concentrations ranging from 0-50 g/L. StartingOD₆₀₀ ranged from 0.03-0.08. Cultures were sparged with CO₂ for 10seconds, sealed, and incubated at 30 C for about 24 hours. OD₆₀₀ wasrecorded every 1-2 hours for the first 8-12 hours with a final OD₆₀₀recorded at about 24 hours. For each data point the sample was opened,sampled, re-sparged with CO₂, and sealed once again. Maximum specificgrowth rates (μ_(max)) were calculated by determining the optimal fit ofexponential trend lines with OD data for the evaluation period. Specificchanges in OD₆₀₀ over approximately 24 hours (Δ_(24 hr)OD₆₀₀) werecalculated as the difference in t=24 hr and t=0 optical density,Δ_(24 hr)OD₆₀₀=(OD_(24 hr))−(OD₆₀₀). Specific number of doublings(N_(d)) were calculated by solving for N in the equation2^(N)=(OD_(t=24))/(OD₆₀₀).

Example 44 Genetic Modification by Introduction of Genes Identified asable to Increase Microorganism Tolerance to 3-HP

Genetic elements containing one to several genes have been identified bythe SCALES 3-HP tolerance data as important to 3-HP tolerance. In orderto develop an optimal combination of these elements suitable toimparting greater tolerance on an organism, a number of these geneticelements have been cloned into a series of compatible plasmidscontaining different origins of replication and selection markers. Assuch, combinations of these compatible plasmids can be transformed intocell lines in order to assess a combinatorial affect on 3-HP tolerance.The parent plasmid vectors containing the different origins ofreplication and selection markers are identified in the following table,which provides SEQ ID numbers (SEQ NOs:005-012 and 183-186) for eachsuch parent plasmid vectors. These plasmids were used to construct theplasmids described herein, and these plasmids, without insert, were alsoused for constructing control cell lines for tolerance MIC testing.

TABLE 41 Vector Sequence pSMART-HC-Amp SEQID. 005 pSMART-LC-Kan SEQID.006 pBT-3 SEQID. 007 pKK223-3 SEQID. 008 pACYC177 (kan only) SEQID. 009pWH1520 SEQID. 010 pHT08 SEQID. 011 pJ61:25125 SEQID. 012 pYes2.1-topoSEQID. 183 pRS423 SEQID. 184 pRS425 SEQID. 185 pJ251 SEQID. 186

Method A: Plasmid Design and Construction of Toleragenic GeneticElements by Gene Synthesis

A single plasmid comprising a number of identified genetic elements wasconstructed in a manner that a plurality of other plasmids could easilybe constructed (some of which were constructed as described). Theseoperons, including a constitutive E. coli promoter, ribosome bindingsites, and open region frames of these genetic elements, were combinedin the single plasmid, which was produced by the gene synthesis servicesof DNA2.0 (Menlo Park, Calif. USA), a commercial DNA gene synthesisprovider. Each of the open reading frames for producing proteins wascodon optimized according to the services of DNA2.0. Additionally,restriction sites were incorporated between each operon and gene togenerate plasmids capable of expressing all combinations of theseproteins through a series of restriction digests and self ligation.Other features of this constructs include an rmB terminator sequenceafter the final operons and mosaic ends containing AfeI restrictionsites flanking each end of the coding region for use with a EZ::TN™Transposon system obtained from EPICENTRE (Madison, Wis.) for futuregenomic incorporation of these elements into strains. This constructedplasmid was provided in a pJ61 vector backbone. The sequence of theresulting vector, termed pJ61:25135, is provided as SEQ ID NO:012.

By the method described herein various nucleic acid sequences encodingenzymes that catalyze enzymatic conversion steps of the 3HPTGC wereintroduced into the pJ61:25135 plasmid. As shown in the following table,the pJ61:25135 plasmid was variously modified to contain gene optimizedsequences for CynS and CynT expressed under a modified Ptrc promoterlocated between Pm1I and SfoI restriction sites, AroG expressed under aPtpiA promoter located between SfoI and SmaI restriction sites (SEQ IDNO:013), SpeD, SpeE, and SpeF expressed under a modified Ptrc promoterlocated between SmaI and ZraI restriction sites (SEQ ID NO:014), ThrAexpressed under a PtalA promoter located between ZraI and HpaIrestriction sites (SEQ ID NO:015), Asd expressed under a PrpiA promoterlocated between HpaI and PmeI restriction sites (SEQ ID NO:016), CysMexpressed under a Ppgk promoter located between PmeI and ScaIrestriction sites (SEQ ID NO:017), IroK expressed under a PtpiA promoterlocated between ScaI and NaeI restriction sites, and IlvA expressedunder a PtalA promoter located between NaeI and EcoICRI restrictionsites (SEQ ID NO:018). Each of these restriction sites is unique withinthe pJ61:25135 plasmid.

TABLE 42 E. coli Tolerance Plasmid Construction PCR Sequence or Gene(s)or Cloning Codon Optimized Region Name Vector Method Primer A Primer BSequence (Region) Plasmid Name aroG pJ61 A N/A N/A SEQID 0013 pJ61-aroGspeFED pJ61 A N/A N/A SEQID 0014 pJ61-speFED thrA pJ61 A N/A N/A SEQID0015 pJ61-thrA Asd pJ61 A N/A N/A SEQID 0016 pJ61-asd cysM pJ61 A N/AN/A SEQID 0017 pJ61-cysM ilvA pJ61 A N/A N/A SEQID 0018 pJ61-ilvA aroHpKK223 B N/A N/A N/A pKK223-aroH aroH G149C pKK223 B N/A N/A N/ApKK223-aroH*445 aroH G149D pKK223 B N/A N/A N/A pKK223-aroH*447 aroHP18L pKK223 B N/A N/A N/A pKK223-aroH*457 metE C645A pKK223 B N/A N/AN/A pKK223-metE C645A thrA pKK223 B N/A N/A SEQIC 0019 pKK223-thrA cynTSpSMART-LC-Kan B N/A N/A SEQIC 0020 pSmart-LC-Kan-cynTS folA C1pSMART-LC-KAN C SEQID 0021 SEQID 0022 SEQID 0023 pSmart-LC-Kan-folA-C1folA ORF pSMART-LC-KAN C SEQID 0024 SEQID 0025 SEQID 0026pSmart-LC-Kan-folA-ORF folD pSMART-LC-KAN C SEQID 0027 SEQID 0028 SEQID0029 pSmart-LC-Kan-folD aroKB C1 pSMART-LC-KAN C SEQID 0030 SEQID 0031SEQID 0032 pSmart-LC-Kan-aroKB C1 pheA C1 pSMART-LC-KAN C SEQID 0033SEQID 0034 SEQID 0035 pSmart-LC-Kan-pheA C1 pheA C2 pSMART-LC-KAN CSEQID 0036 SEQID 0037 SEQID 0038 pSmart-LC-Kan-pheA C2 menA C1pSMART-LC-KAN C SEQID 0039 SEQID 0040 SEQID 0041 pSmart-LC-Kan-menA C1menA ORF pSMART-LC-KAN C SEQID 0042 SEQID 0043 SEQID 0044pSmart-LC-Kan-menA ORF serA pSMART-LC-KAN C SEQID 0045 SEQID 0046 SEQID0047 pSmart-LC-Kan-serA glyA C1 pSMART-LC-KAN C SEQID 0048 SEQID 0049SEQID 0050 pSmart-LC-Kan-glyA C1 glyA ORF pSMART-LC-KAN C SEQID 0051SEQID 0052 SEQID 0053 pSmart-LC-Kan-glyA ORF metC C1 pSMART-LC-KAN CSEQID 0054 SEQID 0055 SEQID 0056 pSMART-LC-KAN-metC C1 tyrApSMART-LC-KAN C SEQID 0057 SEQID 0058 SEQID 0059 pSmart-LC-Kan-tyrA tyrA-aroF pSMART-LC-KAN C SEQID 0060 SEQID 0061 SEQID 0062pSmart-LC-Kan-tyrA-aroF aroE pSMART-LC-KAN C SEQID 0063 SEQID 0064 SEQID0065 pSmart-LC-Kan-aroE ilvA pSMART-LC-KAN C SEQID 0066 SEQID 0067 SEQID0068 pSmart-LC-KAN-ilvA C1 ilvA pSMART-LC-KAN C SEQID 0069 SEQID 0070SEQID 0071 pSmart-LC-KAN-ilvA operon cysM pSMART-LC-KAN C SEQID 0072SEQID 0073 SEQID 0074 pSmart-LC-Kan-cysM cynTS pSMART-HC-AMP D SEQID0075 SEQID 0076 SEQID 0077 pSmart-HC-Amp-cynTS metC pSMART-HC-Amp DSEQID 0078 SEQID 0079 SEQID 0080 pSmart-HC-Amp-metC dapA pSMART-HC-Amp ESEQID 0081* SEQID 0082* SEQID 0083 pSmart-HC-Amp-dapA cadA pSMART-HC-AmpE SEQID 0084* SEQID 0085* SEQID 0086 pSmart-HC-Amp-cadA prspSMART-HC-Amp E SEQID 0087* SEQID 0088* SEQID 0089 pSmart-HC-Amp-prsnrdAB pSMART-HC-Amp E SEQID 0090* SEQID 0091* SEQID 0092pSmart-HC-Amp-nrdAB nrdLEF pSMART-HC-Amp E SEQID 0093* SEQID 0094* SEQID0095 pSmart-HC-Amp-nrdLEF lysA pSMART-HC-Amp E SEQID 0096* SEQID 0097*SEQID 0098 pSMART-HC-Amp-lysA cyntTS pACYC177 (kan only) F SEQID 0099SEQID 0100 SEQID 0101 pACYC177-cynTS aroH G149C pACYC177 (kan only) FSEQID 0102 SEQID 0103 SEQID 0104 pACYC177-aroH* speB pACYC177 (kan only)F SEQID 0105 SEQID 0106 SEQID 0107 pACYC177-speB metE C645A pACYC177(kan only) F SEQID 0108 SEQID 0109 SEQID 0110 pACYC177-metE* metCpACYC177 (kan only) F SEQID 0111 SEQID 0112 SEQID 0113 pACYC177-metCcyntTS pBT-3 G SEQID 0114 SEQID 0115 SEQID 0116 pBT-3-cynTS aroH G149CpBT-3 G SEQID 0117 SEQID 0118 SEQID 0119 pBT-3-aroH* speB pBT-3 G SEQID0120 SEQID 0121 SEQID 0122 pBT-3-speB *5′phosphorylated

To create a set of plasmids containing each of these single operons, aseries of restrictions and self-ligations are performed. As such, anyoperons can be isolated by removal of the DNA sequences between itsflanking restriction sites and the EcoICRI and PmII sites flanking theentire protein coding region of the plasmid. For example, the plasmidcomprising the operon comprising the AroG polypeptide, expressed under aPtpiA promoter and located between SfoI and SmaI restriction sites, wascreated by first digesting the pJ61:25135 plasmid with PmII and SfoIobtained from New England BioLabs (Ipswich, Mass. USA) according tomanufacturer's instructions. The resulting DNA was then self-ligatedwith T4 DNA ligase obtained front New England BioLabs (Ipswich, Mass.USA) according to manufacturer's instructions, and transformed into E.coli K12. Individual colonies from this E. coli K12 transformation weregrown in liquid culture and plasmids from individual colonies wereisolated using a Qiagen Miniprep kit (Valencia, Calif. USA) according tomanufacturer's instructions, The isolated plasmids were screened byrestriction digests with AfeI, and correct plasmids were carried on thenext round of restriction and self ligation. In the second round, theseplasmids were subjected to restriction with SmaI and EcoICRI obtainedfrom New England BioLabs (Ipswich, Mass. USA) and Promega Corporation(Madison, Wis.), respectively, according to manufacturer's instructions.The resulting DNA was then self-ligated with T4 DNA ligase obtained fromNew England BioLabs (Ipswich, Mass. USA) according to manufacturer'sinstructions, and transformed into E. coli K12. Individual colonies fromthis E. coli K12 transformation were grown in liquid culture andplasmids from individual colonies were isolated using a Qiagen Miniprepkit (Valencia, Calif. USA) according to manufacturer's instructions. Theisolated plasmids were screened by restriction digests with AfeI, andverified by sequencing.

In a similar manner using the corresponding restriction sites listedabove the following plasmids were created: pJ61-IlvA expressed under aPtalA promoter located between NaeI and EcoICRI restriction sites;pJ61-CysM expressed under a Ppgk promoter located between PmeI and ScaIrestriction sites; pJ61 -Asd expressed under a PrpiA promoter locatedbetween HpaI and PmeI restriction sites; pJ61-ThrA expressed under aPtalA promoter located between ZraI and HpaI restriction sites;pJ61-SpeDEF expressed under a Ptrc promoter located between SmaI andZraI restriction sites; pJ61-AroG expressed under a PtpiA promoterlocated between SfoI and SmaI restriction sites; and pJ61-CynTSexpressed under a Ptrc promoter located between Pm1I and SfoIrestriction sites. Likewise, any combination of these operons can beobtained via a similar restriction and self-ligation scheme.

These sequence-verified plasmids were transformed into BW25113 E. colicells as tested for tolerance to 3-HP. In addition, these plasmids canbe restricted with AfeI and the purified piece containing the individualoperons with mosaic ends can be incorporated into the genome of a cellline using, the EZ::TN™ Transposon system obtained from EPICENTRE(Madison, Wis.) using the manufactures instructions. Likewise, theseoperons can be moved to any variety of plasmids from providingadditional control of expression or for propagation in a variety ofstrains or organisms.

Method B: Plasmid Containing Identified Elements Received from OtherLabs

After development of the map of the 3HPTGC, a literature reviewidentified previous work on several of the identified genes. Requestswere made to the laboratories that made these reports for plasmidscontaining either the wild-type or mutated genes for the elementsidentified in the 3HPTGC. The so-obtained gene and the proteins theyencode are identified by sequence numbers.

Plasmids containing the wild-type aroH gene and aroH mutants were kindlyprovided as a gift from the Battelle laboratory at the University ofVirginia. These mutants were described in Ray J M, Yanofsky C, BauerleR., J Bacteriol. 1988 December; 170(12);5500-6. Mutational analysis ofthe catalytic and feedback sites of the tryptophan-sensitive3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichiacoli. Along with a pKK223 plasmid containing the wild-type gene, threeadditional pKK223 plasmids were provided containing mutated genes codingfor a glycine to cysteine mutation at position 149, a glycine toaspartic acid mutation at position 149, and a prolific to leucinemutation at position 18.

A plasmid containing a mutant metE gene was kindly provided as a giftfrom the Matthews laboratory at the University of Michigan. This mutantwas described in Hondorp E R, Matthews R G. J Bacteriol. 2009 May;191(10):3407-10. Epub 2009 Mar. 13. Oxidation of cysteine 645 ofcobalamin-independent methionine synthase causes a methionine limitationin Escherichia coli. This pKK233 plasmid carries a metE gene coding fora mutation of a cysteine to an alanine at position 645.

The sequences for the encoded proteins for these genes are provided asSEQ ID NOs:022 to 026.

Method C: Tolerance plasmids construction in a pSMART-LC-Kan vector

Several of the genetic elements that were assessed for their affects on3-HP tolerance were constructed in a pSMART-LC-kan vector (SEQ IDNO:027) obtained from Lucigen Corporation (Middleton Wis., USA). Thisvector provides a low copy replication origin and kanamycin selection.All of these plasmids were created in a similar method and theintroduced genetic elements and the proteins they encode are identifiedby sequence numbers in Table 42 under the method C section therein. Eachrow in Table 42, under method C, contains the respective sequenceinformation for the protein contained within the cloned plasmid, theprimers used in any polymerase chain reactions, and the sequence of thepolymerase chain reaction product used to create the new plasmid.

In each case, an identical procedure was used to create the finalplasmid. The primers listed were used to amplify the correct insertusing pfx DNA polymerase from Invitrogen Corporation (Carlsbad, Calif.USA) and genomic E. coli K12 DNA as template using the manufacturer'sinstructions. The 5′ termini or the amplified DNA product werephosphorylated using T4 polynucleotide kinase for New England Biolabs(Ipswich, Mass. USA) using the manufacturer's instructions. Theresulting product of this reaction was separated by agarose gelelectrophoresis, and a band of the expected size was isolated bydissecting it from the gel and gel extracting the DNA using a gelextraction kit provided by Qiagen Corporation (Valencia, Calif. USA).The extracted phosphorylated DNA was then blunt-end ligated into thepSMART-LC-Kan vector and transformed into 10 G E. coli cells using themanufacturer's instructions. Transformed cells were allowed to recoverin rich media and then were plated on to LB agar plated containingkanamycin for proper selection. After colony growth, single colonieswere grown in LB media and plasmid DNA was extracted using miniprep kitsobtained from Qiagen Corporation (Valencia, Calif. USA). The isolatedplasmid DNA was checked by restriction digest and sequenced verifiedbefore use in other experiments.

Method D: Tolerance Plasmids Construction in a pSMART-HC-Amp Vector

Several of the genetic elements that were assessed for their affects on3-HP tolerance were constructed in a pSMART-HC-AMP vector obtained fromLucigen Corporation (Middleton Wis., USA). This vector provides a highcopy replication origin and ampicillin selection. All of these plasmidswere created in a similar method and are identified as method D in table42. Each row in Table 42 contains the sequence information for theprotein contained within the cloned plasmid, the primers used in anypolymerase chain reactions, and the sequence of the polymerase chainreaction product used to create the new plasmid.

In each case, an identical procedure was used to create the finalplasmid. The primers listed were used to amplify the correct insertusing KOD DNA polymerase from EMD Chemical Corporation (Gibbstown, N.J.USA) and the pKK223 plasmids for each corresponding gene or geneticelements created with method B of Table 42 as template using themanufacturer's instructions. The 5′ termini of the amplified DNA productwere phosphorylated using T4 polynucleotide kinase for New EnglandBiolabs (Ipswich, Mass. USA) using the manufacturer's instructions. Theresulting product of this reaction was separated by agarose gelelectrophoresis, and a hand of the expected size was isolated bydissecting it from the gel and gel extracting the DNA using a gelextraction kit provided by Qiagen Corporation (Valencia, Calif. USA).The extracted phosphorylated DNA was then blunt-end ligated into thepSMART-HC-AMP vector and transformed into 10 G E. coli cells using themanufacturer's instructions. Transformed cells were allowed to recoverin rich media and then were plated on to LB agar plated containingampicillin for proper selection. After colony growth, single colonieswere grown in LB media and plasmid DNA was extracted using miniprep kitsobtained from Qiagen Corporation (Valencia, Calif. USA). The isolatedplasmid DNA was checked by restriction digest and sequenced verifiedbefore use in other experiments.

Method E: Additional Tolerance Plasmids Construction in a pSMART-HC-AmpVector

Several of the genetic elements that were assessed for their affects on3-HP tolerance were constructed in a pSMART-HC-AMP vector obtained fromLucigen Corporation (Middleton Wis., USA). This vector provides a highcopy replication origin and ampicillin selection. All of these plasmidswere created in a similar method and are identified as method E in Table42. Each row in Table 42 contains the sequence information for theprotein contained within the cloned plasmid, the primers used in anypolymerase chain reactions, and the sequence of the polymerase chainreaction product used to create the new plasmid.

In each case, an identical procedure was used to create the finalplasmid. The primers listed were used to amplify the correct insertusing KOD DNA polymerase from EMD Chemical Corporation (Gibbstown, N.J.USA) and genomic E. coli K12 DNA as template using the manufacturer'sinstructions. Since the 5′ termini of the primers were alreadyphosphorytated, no other treatment was needed for the amplified product.The resulting product of this reaction was separated by agarose gelelectrophoresis, and a hand of the expected size was isolated bydissecting it from the gel and gel extracting the DNA using a gelextraction kit provided by Qiagen Corporation (Valencia, Calif. USA).The extracted phosphorylated DNA was then blunt-end ligated into thepSMART-HC-Amp vector and transformed into 10G E. coli cells using themanufacturer's instructions. Transformed cells were allowed to recoverin rich media and then were plated on to LB agar plated containingampicillin for proper selection. After colony growth, single colonieswere grown in LB media and plasmid DNA was extracted using miniprep kitsobtained from Qiagen Corporation (Valencia, Calif. USA). The isolatedplasmid DNA was checked by restriction digest and sequenced verifiedbefore use in other experiments.

Method F: Tolerance Plasmids Construction in a pACYC177 (Kan Only)Vector

Several of the genetic elements that were assessed for their affects on3-HP tolerance were constructed in a pACYC177 (Kan only) vector. Thisbackbone was created by amplifying a portion of the pACYC177 plasmidusing the primer CPM0075 (5′-CGCGGTATCATTGCAGCAC-3′) (SEQ NO:123) andprimer CPM0018 (5′-GCATCGGCTCTTCCGCGTCAAGTCAGCGTAA-3′) (SEQ ID NO:124)using KOD polymerase from EMD Chemical Corporation (Gibbstown, N.J.USA). The resulting product of this reaction was separated by agarosegel electrophoresis, and a band of the expected size was isolated bydissecting it from the gel and gel extracting the DNA using a gelextraction kit provided by Qiagen Corporation (Valencia, Calif. USA).This DNA was designated pACYC177 (Kan only) and was kept for ligation tothe products created herein. This pACYC177 (Kan only) backbone DNAprovides low copy replication origin and kanamycin selection. All ofthese plasmids were created in a similar method and are identified asmethod F in Table 42. Each row in Table 42 contains the sequenceinformation for the protein contained within the cloned plasmid, theprimers used in any polymerase chain reactions, and the sequence of thepolymerase chain reaction product used to create the new plasmid.

In each case, an identical procedure was used to create the finalplasmid. The primers listed were used to amplify the correct insertusing KOD DNA polymerase from EMD Chemical Corporation (Gibbstown, N.J.USA) using the manufacturer's instructions with either the pKK223plasmids for each corresponding gene (or genetic element) created withmethod B of Table 42 or with genomic E. coli DNA as template. The 5′termini or the amplified DNA product were phosphorylated using T4polynucleotide kinase for New England Biolabs (Ipswich, Mass. USA) usingthe manufacturer's instructions. The resulting product of this reactionwas separated by agarose gel electrophoresis, and a hand of the expectedsize was isolated by dissecting it from the gel and gel extracting theDNA using a gel extraction kit provided by Qiagen Corporation (Valencia,Calif. USA). The extracted phosphorylated DNA was then blunt-end ligatedto the pACYC177 (Kan only) backbone DNA described herein and transformedinto 10G E. coli cells using the manufacturer's instructions.Transformed cells were allowed to recover in rich media and then wereplated on to LB agar plated containing kanamycin for proper selection.After colony growth, single colonies were grown in LB media and plasmidDNA was extracted using miniprep kits obtained from Qiagen Corporation(Valencia, Calif. USA). The isolated plasmid DNA was checked byrestriction digest and sequenced verified before use in otherexperiments.

Method G: Tolerance Plasmids Construction in a pBT-3 Vector

Several of the genetic elements that were assessed for their affects on3-HP tolerance were constructed in a pBT-3 vector. This backbone wascreated by amplifying a portion of the pBT-3 plasmid using the primerPET-FOR (5′-AACGAATTCAAGCTTGATATC-3′) (SEQ ID NO:125) and primer PBT-REV(5′-GAATTCGTTGACGAATTCTCTAG-3′) (SEQ ID NO:126) using KOD polymerasefrom EMD Chemical Corporation (Gibbstown, N.J. USA). The resultingproduct of this reaction was separated by agarose gel electrophoresis,and a hand of the expected size was isolated by dissecting it from thegel and gel extracting the DNA using a gel extraction kit provided byQiagen Corporation (Valencia, Calif. USA). This DNA was designated pBT-3backbone and was kept for ligation to the products created herein. ThispBT-3 backbone DNA provides low copy replication origin andchloramphenicol selection. All of these plasmids were created in asimilar method and are identified as method G in Table 42. Each row inTable 42 contains the sequence information for the protein containedwithin the cloned plasmid, the primers used in any polymerase chainreactions, and the sequence of the polymerase chain reaction productused to create the new plasmid.

In each case, an identical procedure was used to create the finalplasmid. The primers listed were used to amplify the correct insertusing KOD DNA polymerase from EMD Chemical Corporation (Gibbstown, N.J.USA) using the manufacturer's instructions with either the pKK223plasmids for each corresponding gene (or genetic element) created withmethod B of Table 42 or with genomic E. coli DNA as template. The 5′termini or the amplified DNA product were phosphorylated using T4polynucleotide kinase for New England Biolabs (Ipswich, Mass. USA) usingthe manufacturer's instructions. The resulting product of this reactionwas separated by agarose gel electrophoresis, and a band of the expectedsize was isolated by dissecting it front the gel and gel extracting theDNA using a gel extraction kit provided by Qiagen Corporation (Valencia,Calif. USA). The extracted phosphorylated DNA was then blunt-end ligatedto the pBT-3 backbone DNA described herein and transformed into 10G E.coli cells using the manufacturer's instructions. Transformed cells wereallowed to recover in rich media and then were plated on to LB agarplated containing chloramphenicol for proper selection. After colonygrowth, single colonies were grown in LB media and plasmid DNA wasextracted using miniprep kits obtained from Qiagen Corporation(Valencia, Calif. USA). The isolated plasmid DNA was checked byrestriction digest and sequenced verified before use in otherexperiments.

Example 45 Evaluation of a Novel Peptide Related to 3-HP Tolerance

A novel 21 amino acid peptide, termed IroK, has been discovered thatincreases 3-HP tolerance.

Methods: IroK Expression Studies

Primers including the entire IroK polypeptide region and RBS flanked byEcorI and HindIII restriction sites were obtained for expression studies(Operon, Huntsville, Ala.):

(SEQ ID NO: 127) (5′-AATTCGTGGAAGAAAGGGGAGATGAAGCCGCTCATTACGCGATTTCATCGCCATTGTGCAGGAACGTTTGGCAACCGTAACGGCATAA-3′, (SEQ ID NO: 128)5′-AGCTTTATGCCGTTACGCTTGCCAAACGTTCCTGCACAATGGCGATGAAATCGCGTAATGCCGGCTTCATCTCCCCTTTCTTCCACG-3′)

Primers including the IroK peptide region and RBS with a mutated startsite (ATG to TTG) were used for the translational analysis:

(SEQ ID NO: 187) (5′-AATTCGTGGAAGAAAGGGGAGTTGAAGCCGGCATEACGCGATTTCATCGCCATTGTGCAGGAACGTTTCTGCAAGCGTAACGGCATAA-3′, (SEQ ID NO: 188)5′-AGCTTTATGCCGTTACGCTTGCCAAACGTTCCTGCACAATGGCGATGAAATCGCGTAATGCCGGCTTCAACTCCCCTTTCTTCCACG-3′)

The two oligonucleotides were added in a 1:1 ratio and annealedaccording to standard methodology in a thermal cycler. Ligation of theannealed primer product with the pKK223-3 expression vector (SEQ IDNO:008, Pharmacia, Piscataway, N.J.) was performed with T4 Ligase(Invitrogen, Carlsbad, Calif.) and incubated at 25° C. overnight. Theligation product was then electroporated into competent MACH1-T1®,plated on LB fampicillan, and incubated at 37° C. for 24 hours. Plasmidswere isolated and confirmed by purification and subsequent restrictiondigest and sequencing (Macrogen, Rockville, Md.). MICS were thendetermined corresponding to 1 mM IPTG induction.

Minimum Inhibitory Concentrations (MIC)

The minimum inhibitory concentration (MIC) was determinedmicroaerobically in a 96 well-plate format. Overnight cultures ofstrains were grown in 5 mL LB (with antibiotic where appropriate). A 1%(v/v) inoculum was introduced into a 15 ml culture of MOPS minimalmedia. After the cells reached mid-exponential phase, the culture wasdiluted to an OD₆₀₀ of 0.200. The cells were further diluted 1:20 and a10 μL aliquot was used to inoculate each well of a 96 well plate (˜10⁴cells per well). The plate was arranged to measure the growth ofvariable strains or growth conditions in increasing 3-HP concentrations,0 to 70 g/L, in 5 g/L increments, The minimum inhibitory 3-HPconcentration and maximum 3-HP concentration corresponding to visiblecell growth (OD˜0.1) was recorded after 24 hours.

Results

To explore the effects of IroK, a peptide comprised of 21 amino acids(MKPALRDFIAIVQERLASVTA, SEQ ID NO:129), the sequence encoding for italong with the native predicted RBS was incorporated into an inducibleexpression vector (pKK223-3). FIG. 20 shows increased expression of theshort 87 bp sequence which is sufficient to enhance tolerance to 3-HP(>2 fold increase in MIC). Additionally, the tolerance mechanism appearsto be specific to 3-HP growth inhibition, as MICs remained unchanged forseveral other organic acids of similar molecular makeup includinglactic, acrylic, and acetic acids. In an effort to dissect the mode oftolerance conferred, a nearly identical sequence was incorporated intothe same vector with a single mutation in the translational start site(ATG to TTG), resulting in a decreased MIC equivalent to that ofwild-type E. coli. (FIG. 20). This result implies that the mechanism oftolerance is specific to the expression of the translated polypeptiderather than mapped to the DNA or RNA level.

A nucleic acid sequence encoding the IroK peptide, or suitable variantsof it, may be provided to a microorganism, that may comprise one or moregenetic modifications of the 3HPTGC to further increase 3-HP tolerance,and that also may have 3-HP production capability.

Example 46 Genetic Modification/Introduction of Malonyl-CoA Reductasefor 3-HP Production in E. coli DF40

The nucleotide sequence for the malonyl-coA reductase gene fromChloroflexus aurantiacus was codon optimized for E. coli according to aservice from DNA 2.0 (Menlo Park, Calif. USA), a commercial DNA genesynthesis provider. This gene sequence incorporated an EcoRI restrictionsite before the start codon and was followed by a HindIII restrictionsite. In addition a Shine Delgarno sequence (i.e., a ribosomal bindingsite) was placed in front of the start codon preceded by an EcoRIrestriction site. This gene construct was synthesized by DNA 2.0 andprovided in a pJ206 vector backbone. Plasmid DNA pJ206 containing thesynthesized mcr gene was subjected to enzymatic restriction digestionwith the enzymes EcoRI and HindIII obtained from New England BioLabs(Ipswich, Mass. USA) according to manufacturer's instructions. Thedigestion mixture was separated by agarose gel electrophoresis, andvisualized under UV transillumination as described in Subsection II ofthe Common Methods Section. An agarose gel slice containing a DNA piececorresponding to the mcr gene was cut from the gel and the DNA recoveredwith a standard gel extraction protocol and components from Qiagen(Valencia, Calif. USA) according to manufacturer's instructions. An E.coli cloning strain bearing pKK223-aroH was obtained as a kind a giftfrom the laboratory of Prof. Ryan T. Gill from the University ofColorado at Boulder. Cultures of this strain bearing the plasmid weregrown by standard methodologies and plasmid DNA was prepared by acommercial mini prep column from Qiagen (Valencia, Calif. USA) accordingto manufacturer's instructions. Plasmid DNA was digested with therestriction endonucleases EcoRI and HindIII obtained from New EnglandBiolabs (Ipswich, Mass. USA) according to manufacturer's instructions.This digestion served to separate the aroH reading frame from the pKK223backbone. The digestion mixture was separated by agarose gelelectrophoresis, and visualized under UV transillumination as describedin Subsection II of the Common Methods Section. An agarose gel slicecontaining a DNA piece corresponding to the backbone of the pKK223plasmid was cut from the gel and the DNA recovered with a standard gelextraction protocol and components from Qiagen according tomanufacturer's instructions.

Pieces of purified DNA corresponding to the mcr gene and pKK223 vectorbackbone were ligated and the ligation product was transformed andelectroporated according to manufacturer's instructions. The sequence ofthe resulting vector termed pKK223-mcr (SEQ ID NO:189) was confirmed byroutine sequencing performed by the commercial service provided byMacrogen (USA). pKK223-mcr confers resistance to beta-lactamase andcontains mcr gene under control of a Ptac promoter inducible in E. colihosts by IPTG.

The expression clone pKK223-mcr and pKK223 control were transformed intoboth E. coli K12 and E. coli DF40 via standard methodologies. (Sambrookand Russell, 2001).

Example 47 Construction of E. coli Gene Deletion Strains

The following strains were obtained from the Keio collection: JW 1650(ΔpurR), JW2807 (ΔlysR), JW1316 (ΔtyrR), JW4356 (ΔtrpR), JW3909 (ΔmetJ).JW0403 (ΔnrdR). The Keio collection was obtained from Open Biosystems(Huntsville, Ala. USA 35806). Individual clones may be purchased fromthe Yale Genetic Stock Center (New Haven, Conn. USA 06520). Thesestrains each contain a kanamycin marker in place of the deleted gene.For more information concerning the Keio Collection and the curing ofthe kanamycin cassette please refer to: Baba, T et al (2006).Construction of Escherichia coli K12 in-frame, single-gene knockoutmutants: the Keio collection. Molecular Systems Biologydoi:10.1038/msb4100050 and Datsenko K A and B L Wanner (2000). One-stepinactivation of chromosomal genes in Escherichia coli K-12 using PCRproducts. PNAS 97, 6640-6645. These strains were made electro-competentby standard methodologies. Each strain was then transformed via standardelectroporation methods with the plasmid pCP20, which was a kind giftfrom Dr. Ryan Gill (University of Colorado, Boulder, Colo. USA).Transformations were plated on Luria Broth agar plates containing 20μg/mL chloramphenicol and 100 μg/mL ampicillin and incubated for 36hours at 30 degrees Celsius. Clones were isolated from thesetransformation and grown overnight in 10 mL of M9 media lacking anyantibiotics. Colonies were isolated from these cultures by streaking,onto Luria Broth agar plates lacking any antibiotics. Colonies wereconfirmed to have lost the kanamycin marker as well as the plasmid pCP20by confirming, no growth on Luria broth agar plates containing theantibiotics, kanamycin (20 μgl/mL), chloramphenicol (20 μg/mL) andampicillin (100 μg/mL). Isolated clones were confirmed by colony PCR tohave lost the kanamycin cassette. PCRs were carried out using EconoTaqPLUS GREEN 2X master PCR mix, obtained from Lucigen, (Catalog #30033)(Middleton, Wis. USA). PCRs were carried out using a 96 well gradientROBOcycler (Stratagene, La Jolla, Calif. USA 92037) with the followingcycles: 1) 10 min at 95 degrees Celsius, 2) 30 of the following cycles,a) 1 min at 95 degrees Celsius, b) 1 min at 52 degrees Celsius, b) 2 minat 72 degrees Celsius, followed by 3) 1 cycle of 10 minutes at 72degrees Celsius. The Primers used for the PCRs to confirm the removal ofthe kanamycin cassette for each of the clones are given in the followingtable. Primers were purchased front integrated DNA Technologies(Coralville, Iowa USA). The resulting cured strains, called BX_00341.0,BX_00342.0, BX_00345.0, BX_00346.0, BX_00348.0 and BX_00349.0,correspond to JW1316 (ΔtyrR), JW4356 (ΔtrpR), JW3909 (ΔmetJ), JW1650(ΔpurR), JW2807 (ΔlysR) and JW0403 (ΔnrdR) respectively.

TABLE 43 Keio Clone Number Gene Deletion Forward Primer Reverse PrimerJW1650 purR SEQ ID: 130 SEQ ID: 131 JW2807 lysR SEQ ID: 132 SEQ ID: 133JW1316 tyrR SEQ ID: 134 SEQ ID: 135 JW4356 trpR SEQ ID: 136 SEQ ID: 137JW3909 metJ SEQ ID: 138 SEQ ID: 139 JW0403 nrdR SEQ ID: 140 SEQ ID: 141

Example 48 E. coli Strain Construction

According to the respective combinations in Tables 44 and 45, plasmidswere introduced into respective base strains. Alt plasmids wereintroduced at the same time via electroporation using standard methods.Transformed cells were grown on the appropriate media with antibioticsupplementation and colonies were selected based on their appropriategrowth on the selective media.

TABLE 44 E. coli Genetic Modification Results under Aerobic ConditionsMIC % Chromosomal Vector based Toler- Assay MIC Increase Genetic Geneticance Result P- Assay Over Strain Name Media (M9+) Parent ModificationsModifications Group (g/L 3-HP) value Number Control BX_00138.0 Kan (20μg/mL) BW25113 wild type pSmart-LC-Kan None 25 <0.1 ≧3 — BX_00300.0 Kan20 μg/mL BW25113 wild type pSmart-LC-Kan- A 35 <0.1 ≧3 40 tyrA-aroFBX_00301.0 Kan 20 μg/mL BW25113 wild type pSmart-LC-Kan- A 35 <0.1 ≧3 40folA-C1 BX_00302.0 Kan 20 μg/mL BW25113 wild type pSmart-LC-Kan- A 30<0.1 ≧3 20 folA-ORF BX_00304.0 Kan 20 μg/mL BW25113 wild typepSmart-LC-Kan- A 35 <0.1 ≧3 40 menA-ORF BX_00305.0 Kan 20 μg/mL BW25113wild type pSmart-LC-Kan- A 35 <0.1 ≧3 40 pheA-C1 BX_00307.0 Kan 20 μg/mLBW25113 wild type pSmart-LC-Kan- A 35 <0.1 ≧3 40 tyrA-C1 BX_00309.0 Kan20 μg/mL BW25113 wild type pSmart-LC-Kan- C 35 <0.1 ≧3 40 cynTSBX_00310.0 Kan 20 μg/mL BW25113 wild type pSmart-LC-Kan- B 35 <0.1 ≧3 40glyA BX_00312.0 Kan 20 μg/mL BW25113 wild type pSmart-LC-Kan- B 35 <0.1≧3 40 serA BX_00313.0 Kan 20 μg/mL BW25113 wild type pSmart-LC-Kan- A 30<0.1 ≧3 20 folD BX_00314.0 Kan 20 μg/mL BW25113 wild type pSmart-LC-Kan-A 35 <0.1 ≧3 40 aroE BX_00315.0 Kan 20 μg/mL BW25113 wild typepSmart-LC-Kan- A 35 <0.1 ≧3 40 aroKB C1 BX_00317.0 Kan 20 μg/mL BW25113wild type pSmart-LC-Kan- B 35 <0.1 ≧3 40 ilvA operon BX_00318.0 Kan 20μg/mL BW25113 wild type pSmart-LC-Kan- B 35 <0.1 ≧3 40 cysM BX_00352.0Amp 100 μg/mL BW25113 wild type pSmart-LC-Kan- B 35 <0.1 ≧3 40 metC C1BX_00387.0 Kan (20 μg/mL) BW25113 ΔlysR::frt pSmart-LC-Kan- A 35 <0.1 ≧340 menA-ORF BX_00002.0 Amp (100 μg/mL) BW25113 wild type pKK223-mcs1None 20 <0.1 ≧3 — BX_00319.0 Amp 100 μg/mL + BW25113 wild typepK223-aroH A 30 <0.1 ≧3 50 1 mM IPTG BX_00320.0 Amp 100 μg/mL + BW25113wild type pK223-metE C645A B 35 <0.1 ≧3 75 1 mM IPTG BX_00321.0 Amp 100μg/mL + BW25113 wild type pK223-ct-his-thrA B 35 <0.1 ≧3 75 1 mM IPTGBX_00357.0 Amp 100 μg/mL + BW25113 wild type pKK223-aroH*445 A 30 <0.1≧3 50 1 mM IPTG BX_00358.0 Amp 100 μg/mL + BW25113 wild typepKK223-aroH*447 A 35 <0.1 ≧3 75 1 mM IPTG BX_00359.0 Amp 100 μg/mL +BW25113 wild type pKK223-aroH*457 A 35 <0.1 ≧3 75 1 mM IPTG BX_00118.0Kan(20 μg/mL) BW25113 wild type pJ251 None 25 <0.1 ≧3 — BX_00322.0 Kan20 μg/mL BW25113 wild type pJ61-speFED C 35 <0.1 ≧3 40 BX_00323.0 Kan 20μg/mL BW25113 wild type pJ61-aroG A 35 <0.1 ≧3 40 BX_00324.0 Kan 20μg/mL BW25113 wild type pJ61-thrA B 35 <0.1 ≧3 40 BX_00325.0 Kan 20μg/mL BW25113 wild type pJ61-asd B 35 <0.1 ≧3 40 BX_00326.0 Kan 20 μg/mLBW25113 wild type pJ61-ilvA B 35 <0.1 ≧3 40 BX_00327.0 Kan 20 μg/mLBW25113 wild type pJ61-cysM B 35 <0.1 ≧3 40 BX_00361.0 Kan 20 μg/mLBW25113 wild type pACYC177 (Kan C 35 <0.1 ≧3 40 only) - cynTS BX_00362.0Kan 20 μg/mL + BW25113 wild type pACYC177 (Kan A 30 <0.1 ≧3 20 1 mM IPTGonly) - aroH BX_00363.0 Kan 20 μg/mL BW25113 wild type pACYC177 (kan C35 <0.1 ≧3 40 only) - speB BX_00364.0 Kan 20 μg/mL + BW25113 wild typepACYC177 (Kan B 35 <0.1 ≧3 40 1 mM IPTG only) - metE (Version1)(SS090608_13) BX_00365.0 Kan 20 μg/mL BW25113 wild type pACYC177 (Kan B35 <0.1 ≧3 40 only) -metC (Version1) (SS090608_17) BX_00144.0 Amp (100μg/mL) BW25113 wild type pSmart-HC-Amp None 25 <0.1 ≧3 — BX_00334.0 Amp100 μg/mL BW25113 wild type pSmart-HC-Amp- D 40 <0.1 ≧3 60 cadABX_00335.0 Amp 100 μg/mL BW25113 wild type pSmart-HC-Amp- E 35 <0.1 ≧340 prs BX_00336.0 Amp 100 μg/mL BW25113 wild type pSmart-HC-Amp- E 35<0.1 ≧3 40 nrdAB BX_00337.0 Amp 100 μg/mL BW25113 wild typepSmart-HC-Amp- E 35 <0.1 ≧3 40 nrdEF BX_00353.0 Amp 100 μg/mL BW25113wild type pSmart-HC-Amp- B 45 <0.1 ≧3 80 metC BX_00354.0 Amp 100 μg/mLBW25113 wild type pSmart-HC-Amp- C 45 <0.1 ≧3 80 cynTS BX_00356.0 Amp100 μg/mL BW25113 wild type pSmart-HC-Amp- D 30 <0.1 ≧3 20 LysABX_00419.0 Amp (100 μg/mL) BW25113 ΔlysR::frt pSmart-HC-Amp- D, E 30<0.1 ≧3 20 prs BX_00420.0 Amp (100 μg/mL) BW25113 ΔlysR::frtpSmart-HC-Amp- D, E 45 <0.1 ≧3 80 nrdAB BX_00421.0 Amp (100 μg/mL)BW25113 ΔlysR::frt pSmart-HC-Amp- D, E 30 <0.1 ≧3 20 nrdEF BX_00425.0Amp (100 μg/mL) BW25113 ΔnrdR::frt pSmart-HC-Amp- D, E 35 <0.1 ≧3 40dapA BX_00426.0 Amp (100 μg/mL) BW25113 ΔnrdR::frt pSmart-HC-Amp- D, E45 <0.1 ≧3 80 cadA BX_00437.0 Amp (100 μg/mL) BW25113 ΔlysR::frtpSmart-HC-Amp- B, D 30 <0.1 ≧3 20 metC BX_00438.0 Amp (100 μg/mL)BW25113 ΔnrdR::frt pSmart-HC-amp- B, D 35 <0.1 ≧3 40 metC BW25113 M9none none none None 27.5 <0.1 ≧3 — BX_00341.0 none BW25113 ΔtyrR::frtnone A 40 <0.1 ≧3 45 BX_00342.0 none BW25113 ΔtrpR::frt none A 35 <0.1≧3 27 BX_00345.0 none BW25113 ΔmetJ::frt none B 35 <0.1 ≧3 27 BX_00347.0none BW25113 ΔpurR::frt none C 35 <0.1 ≧3 27 BX_00348.0 none BW25113ΔlysR::frt none D 35 <0.1 ≧3 27 BX_00349.0 none BW25113 ΔnrdR::frt noneE 35 <0.1 ≧3 27 BX_00003.0 Cm(20 μg/mL) BW25113 wild type pBT-3 None 25<0.1 ≧3 — BX_00368.0 Cm (20 μg/mL) BW25113 wild type pBT-3-cynTS C 30<0.1 ≧3 20 BX_00370.0 Cm (20 μg/mL) BW25113 wild type pBT-3-speB C 30<0.1 ≧3 20 BX_00142.0 Kan(20 μg/mL), BW25113 wild type pSmart-LC-kan,None 20 <0.1 ≧3 — Cm(20 μg/mL) pBT-3 BX_00463.0 Cm (20 μg/mL)/ BW25113ΔnrdR::frt pBT-3-aroH*, A, C, E 30 <0.1 ≧3 50 Kan(20 μg/mL) +pSmart-LC-Kan 1 mM IPTG cynTS BX_00468.0 Cm (20 μg/mL)/ BW25113ΔnrdR::frt pSmart-LC-Kan- B, C, E 30 <0.1 ≧3 50 Kan(20 μg/mL) metC,pBT3-cynTS

TABLE 45 E. coli Genetic Modification Results under Anaerobic ConditionsMIC % Chromosomal Vector based Toler- Assay MIC Increase Genetic Geneticance Result P- Assay Over Strain Name Media (M9+) Parent ModificationsModifications Group (g/L 3-HP) value Number Control BX_00138.0 Kan (20μg/mLl) BW25113 wild type pSmart-LC-Kan None 25 <0.1 ≧3 — BX_00311.0 Kan20 μg/mL BW25113 wild type pSmart-LC-Kan- B 30 <0.1 ≧3 20 glyA-ORFBX_00002.0 Amp (100 μg/mL) BW25113 wild type pKK223-mcs1 None 15 <0.1 ≧3— BX_00319.0 Amp 100 μg/mL + BW25113 wild type pK223-aroH A 20 <0.1 ≧333 1 mM IPTG BX_00320.0 Amp 100 μg/mL + BW25113 wild type pK223-metE B20 <0.1 ≧3 33 1 mM IPTG C645A BX_00321.0 Amp 100 μg/mL + BW25113 wildtype pK223-ct-his-thrA B 20 <0.1 ≧3 33 1 mM IPTG BX_00357.0 Amp 100μg/mL + BW25113 wild type pKK223-aroH*445 B 20 <0.1 ≧3 33 1 mM IPTGBX_00358.0 Amp 100 μg/mL + BW25113 wild type pKK223-aroH*447 A 20 <0.1≧3 33 1 mM IPTG BX_00359.0 Amp 100 μg/mL + BW25113 wild typepKK223-aroH*457 A 20 <0.1 ≧3 33 1 mM IPTG BX_00118.0 Kan(20 μg/mL)BW25113 wild type pJ251 None 15 <0.1 ≧3 — BX_00322.0 Kan 20 μg/mLBW25113 wild type pJ61-speFED C 25 <0.1 ≧3 67 BX_00323.0 Kan 20 μg/mLBW25113 wild type pJ61-aroG A 20 <0.1 ≧3 33 BX_00324.0 Kan 20 μg/mLBW25113 wild type pJ61-thrA B 20 <0.1 ≧3 33 BX_00325.0 Kan 20 μg/mLBW25113 wild type pJ61-asd B 20 <0.1 ≧3 33 BX_00326.0 Kan 20 μg/mLBW25113 wild type pJ61-ilvA B 20 <0.1 ≧3 33 BX_00327.0 Kan 20 μg/mLBW25113 wild type pJ61-cysM B 20 <0.1 ≧3 33 BX_00360.0 Kan 20 μg/mLBW25113 wild type pACYC177(Kan C 20 <0.1 ≧3 33 only)-cynTS BX_00362.0Kan 20 μg/mL + BW25113 wild type pACYC177(Kan A 20 <0.1 ≧3 33 1 mM IPTGonly)-aroH BX_00363.0 Kan 20 μg/mL BW25113 wild type pACYC177(Kan C 20<0.1 ≧3 33 only)-speB BX_00364.0 Kan 20 μg/mL + BW25113 wild typepACYC177(Kan B 20 <0.1 ≧3 33 1 mM IPTG only)-metE BX_00365.0 Kan 20μg/mL BW25113 wild type pACYC177(Kan B 20 <0.1 ≧3 33 only)-metCBX_00144.0 Amp (100 μg/mL) BW25113 wild type pSmart-HC-Amp None 25 <0.1≧3 — BX_00426.0 Amp (100 μg/mL) BW25113 ΔnrdR::frt pSmart-HC-Amp- D, E26.7 <0.1 ≧3  7 cadA BX_00003.0 Cm(20 μg/mL) BW25113 wild type pBT-3None 15 <0.1 ≧3 — BX_00368.0 Cm (20 μg/mL) BW25113 wild type pBT-3-cynTSC 20 <0.1 ≧3 33

Example 49 Evaluation of 3HPTGC-Related Supplements on Wild-Type E. coli

The effect of supplementation on 3HP tolerance was determined by MICevaluations using the methods described in the Common Methods Section.Supplements tested are listed in Table 46. Results of the MICevaluations are provided in Table 47 for aerobic condition and Table 48for anaerobic condition. This data, which includes single andmultiple-supplement additions, demonstrates improvement in 3-HPtolerance in these culture systems based on 24-hour MIC evaluations.

TABLE 46 Supplements TGC Concentration, Supplement Source Group g/L NoteTyrosine Sigma, St. Louis, MO A 0.036 dissolve in 0.01 KOH, pH final to7 Phenylalanine Sigma, St. Louis, MO A 0.0664 Tryptophan Sigma, St.Louis, MO A 0.0208 Shikimate Sigma, St. Louis, MO A 0.1 p-aminobenzoateMP Biomedicals, Aurora, A 0.069 OH Dihydroxybenzoate Sigma, St. Louis,MO A 0.077 Tetrahydrofolate Sigma, St. Louis, MO A 0.015 10% DMSOHomocysteine MP Biomedicals, Aurora, B 0.008 OH Isoleucine Sigma, St.Louis, MO B 0.0052 Serine Sigma, St. Louis, MO B 1.05 Glycine FisherScientific, Fair B 0.06 Lawn, NJ Methionine Sigma, St. Louis, MO B 0.03Threonine Sigma, St. Louis, MO B 0.0476 2-oxobutyrate Fluka Biochemika,B 0.051 Hungary Homoserine Acros Organics, NJ B 0.008 Aspartate Sigma,St. Louis, MO B 0.0684 Putrescine MP Biomedicals, Salon, C 0.9 OHCadaverine MP Biomedicals, Salon, C 0.6 OH Spermidine MP Biomedicals,Salon, C 0.5 OH Ornithine Sigma, St. Louis, MO C 0.2 Citrulline Sigma,St. Louis, MO C 0.2 Bicarbonate Fisher Scientific, Fair C 1 Lawn, NJGlutamine Sigma, St. Louis, MO C 0.09 dissolve in 1M HCl, pH final to 7Lysine Sigma, St. Louis, MO D 0.0732 Uracil Sigma, St. Louis, MO E 0.224Citrate Fisher Scientific, Fair F 2 Lawn, NJ Chorismate Group Mix Seeabove A See respective (includes all Group A concentrations supplementslisted above above) Homocysteine Group See above B See respective Mix(includes all Group concentrations B supplements listed above above)Polyamine Group Mix See above C See respective (includes all Group Cconcentrations supplements listed above above)

TABLE 47 E. coli Supplement Results under Aerobic Conditions average %MIC Assay MIC Increase Result P- Assay Over Strain Name MediaSupplements (Group) (g/L 3-HP) value Number Control CONTROLS BW25113 M9none 28 <0.1 ≧3 — BW25113 EZ Rich none 75 <0.1 ≧3 173 BW25113 M9Phenylalanine (A) 32 <0.1 ≧3 17 BW25113 M9 Shikimate (A) 28 <0.1 ≧3 3BW25113 M9 p-aminobenzoate (A) 35 <0.1 ≧3 27 BW25113 M9Dihydroxybenzoate (A) 35 <0.1 ≧3 27 BW25113 M9 Tetrahydrofolate (A) 30<0.1 ≧3 9 BW25113 M9 Chorismate Group Mix (A) 30 <0.1 ≧3 9 BW25113 M9Homocysteine (B) 30 <0.1 ≧3 9 BW25113 M9 Isoleucine (B) 32 <0.1 ≧3 17BW25113 M9 Serine (B) 32 <0.1 ≧3 17 BW25113 M9 Glycine (B) 28 <0.1 ≧3 3BW25113 M9 Methionine (B) 38 <0.1 ≧3 36 BW25113 M9 Threonine (B) 32 <0.1≧3 17 BW25113 M9 Homoserine (B) 35 <0.1 ≧3 27 BW25113 M9 HomocysteineGroup Mix (B) 40 <0.1 ≧3 45 BW25113 M9 Putrescine(C) 30 <0.1 ≧3 9BW25113 M9 Cadaverine (C) 35 <0.1 ≧3 27 BW25113 M9 Spermidine (C) 40<0.1 ≧3 45 BW25113 M9 Ornithine (C) 30 <0.1 ≧3 9 BW25113 M9 Citrulline(C) 30 <0.1 ≧3 9 BW25113 M9 Bicarbonate (C) 44 <0.1 ≧3 59 BW25113 M9Glutamine(C) 30 <0.1 ≧3 9 BW25113 M9 Polyamine Group Mix (C) 57 <0.1 ≧3106 BW25113 M9 Lysine (D) 37 <0.1 ≧3 33 Double Supplements BW25113 M9Tyrosine (A), Homocysteine (B) 35 <0.1 ≧3 27 BW25113 M9 Tyrosine (A),Methionine (B) 30 <0.1 ≧3 9 BW25113 M9 Tyrosine (A), Isoleucine (B) 30<0.1 ≧3 9 BW25113 M9 Tyrosine (A), Putrescine (C) 40 <0.1 ≧3 45 BW25113M9 Tyrosine (A), Spermidine (C) 40 <0.1 ≧3 45 BW25113 M9 Tyrosine (A),Ornithine (C) 30 <0.1 ≧3 9 BW25113 M9 Tyrosine (A), Bicarbonate (C) 35<0.1 ≧3 27 BW25113 M9 Tyrosine (A), Lysine (D) 30 <0.1 ≧3 9 BW25113 M9Tyrosine (A), Citrate (F) 35 <0.1 ≧3 27 BW25113 M9 Shikimate (A),Methionine (B) 30 <0.1 ≧3 9 BW25113 M9 Shikimate (A), Bicarbonate (C) 30<0.1 ≧3 9 BW25113 M9 Shikimate (A), Uracil (E) 30 <0.1 ≧3 9 BW25113 M9Tetrahydrofolate (A), Methionine (B) 30 <0.1 ≧3 9 BW25113 M9Tetrahydrofolate (A), Homocysteine (B) 30 <0.1 ≧3 9 BW25113 M9Tetrahydrofolate (A), Putrescine (C) 35 <0.1 ≧3 27 BW25113 M9Tetrahydrofolate (A), Spermidine (C) 40 <0.1 ≧3 45 BW25113 M9Tetrahydrofolate (A), Ornithine (C) 35 <0.1 ≧3 27 BW25113 M9Tetrahydrofolate (A), Bicarbonate (C) 30 <0.1 ≧3 9 BW25113 M9Tetrahydrofolate (A), Uracil (E) 30 <0.1 ≧3 9 BW25113 M9Tetrahydrofolate (A), Citrate (F) 30 <0.1 ≧3 9 BW25113 M9 Methionine(B), Putrescine (C) 47 <0.1 ≧3 70 BW25113 M9 Methionine (B), Spermidine(C) 40 <0.1 ≧3 45 BW25113 M9 Methionine (B), Ornithine (C) 45 <0.1 ≧3 64BW25113 M9 Methionine (B), Bicarbonate (C) 35 <0.1 ≧3 27 BW25113 M9Methionine (B), Lysine (D) 30 <0.1 ≧3 9 BW25113 M9 Methionine (B),Uracil (E) 35 <0.1 ≧3 27 BW25113 M9 Methionine (B), Citrate (F) 30 <0.1≧3 9 BW25113 M9 Homocysteine (B), Putrescine (C) 40 <0.1 ≧3 45 BW25113M9 Homocysteine (B), Spermidine (C) 45 <0.1 ≧3 64 BW25113 M9Homocysteine (B), Ornithine (C) 30 <0.1 ≧3 9 BW25113 M9 Homocysteine(B), Bicarbonate (C) 42 <0.1 ≧3 52 BW25113 M9 Homocysteine (B), Lysine(D) 35 <0.1 ≧3 27 BW25113 M9 Homocysteine (B), Uracil (E) 30 <0.1 ≧3 9BW25113 M9 Homocysteine (B), Citrate (F) 30 <0.1 ≧3 9 BW25113 M9Isoleucine (B), Putrescine (C) 35 <0.1 ≧3 27 BW25113 M9 Isoleucine (B),Spermidine (C) 35 <0.1 ≧3 27 BW25113 M9 Isoleucine (B), Bicarbonate (C)35 <0.1 ≧3 27 BW25113 M9 Isoleucine (B), Lysine (D) 30 <0.1 ≧3 9 BW25113M9 Isoleucine (B), Uracil (E) 35 <0.1 ≧3 27 BW25113 M9 Isoleucine (B),Citrate (F) 35 <0.1 ≧3 27 BW25113 M9 Putrescine (C), Lysine (D) 42 <0.1≧3 52 BW25113 M9 Putrescine (C), Uracil (E) 30 <0.1 ≧3 9 BW25113 M9Putrescine (C), Citrate (F) 30 <0.1 ≧3 9 BW25113 M9 Spermidine (C),Lysine (D) 40 <0.1 ≧3 45 BW25113 M9 Spermidine (C), Uracil (E) 30 <0.1≧3 9 BW25113 M9 Spermidine (C), Citrate (F) 38 <0.1 ≧3 39 BW25113 M9Ornithine (C), Lysine (D) 32 <0.1 ≧3 15 BW25113 M9 Ornithine (C), Uracil(E) 30 <0.1 ≧3 9 BW25113 M9 Ornithine (C), Citrate (F) 30 <0.1 ≧3 9BW25113 M9 Bicarbonate (C), Lysine (D) 35 <0.1 ≧3 27 BW25113 M9Bicarbonate (C), Uracil (E) 35 <0.1 ≧3 27 BW25113 M9 Bicarbonate (C),Citrate (F) 40 <0.1 ≧3 45 BW25113 M9 Lysine (D), Uracil (E) 30 <0.1 ≧3 9BW25113 M9 Lysine (D), Citrate (F) 30 <0.1 ≧3 9 Triple SupplementsBW25113 M9 Tyrosine (A), Methionine (B), 35 <0.1 ≧3 27 Putrescine (C)BW25113 M9 Tyrosine (A), Methionine (B), 35 <0.1 ≧3 27 Spermidine (C)BW25113 M9 Tyrosine (A), Methionine (B), 30 <0.1 ≧3 9 Bicarbonate (C)BW25113 M9 Tyrosine (A), Methionine (B), 30 <0.1 ≧3 9 Lysine (D) BW25113M9 Tyrosine (A), Methionine (B), 40 <0.1 ≧3 45 Uracil (E) BW25113 M9Tyrosine (A), Methionine (B), 30 <0.1 ≧3 9 Citrate (F) BW25113 M9Tyrosine (A), Putrescine (C), 30 <0.1 ≧3 9 Homocysteine (B) BW25113 M9Tyrosine (A), Putrescine (C), 28 <0.1 ≧3 3 Isoleucine (B) BW25113 M9Tyrosine (A), Putrescine (C), 35 <0.1 ≧3 27 Lysine (D) BW25113 M9Tyrosine (A), Putrescine (C), 30 <0.1 ≧3 9 Uracil (E) BW25113 M9Tyrosine (A), Spermidine (C), 30 <0.1 ≧3 9 Homocysteine (B) BW25113 M9Tyrosine (A), Spermidine (C), 30 <0.1 ≧3 9 Isoleucine (B) BW25113 M9Tyrosine (A), Spermidine (C), 30 <0.1 ≧3 9 Lysine (D) BW25113 M9Tyrosine (A), Spermidine (C), 35 <0.1 ≧3 27 Uracil (E) BW25113 M9Tyrosine (A), Spermidine (C), 30 <0.1 ≧3 9 Citrate (F) BW25113 M9Tyrosine (A), Bicarbonate (C), 35 <0.1 ≧3 27 Homocysteine (B) BW25113 M9Tyrosine (A), Bicarbonate (C), 35 <0.1 ≧3 27 Isoleucine (B) BW25113 M9Tyrosine (A), Bicarbonate (C), 45 <0.1 ≧3 64 Lysine (D) BW25113 M9Tyrosine (A), Bicarbonate (C), 45 <0.1 ≧3 64 Uracil (E) BW25113 M9Tyrosine (A), Bicarbonate (C), 40 <0.1 ≧3 45 Citrate (F) BW25113 M9Shikimate (A), Putrescine (C), 30 <0.1 ≧3 9 Homocysteine (B) BW25113 M9Shikimate (A), Putrescine (C), 30 <0.1 ≧3 9 Uracil (E) BW25113 M9Shikimate (A), Putrescine (C), 30 <0.1 ≧3 9 Methionine (B) BW25113 M9Shikimate (A), Spermidine (C), 30 <0.1 ≧3 9 Methionine (B) BW25113 M9Shikimate (A), Uracil (C), 30 <0.1 ≧3 9 Homocysteine (B) BW25113 M9Shikimate (A), Uracil (C), 30 <0.1 ≧3 9 Isoleucine (B) BW25113 M9Shikimate (A), Uracil (C), 35 <0.1 ≧3 27 Methionine (B) BW25113 M9Shikimate (A), Uracil (C), 30 <0.1 ≧3 9 Lysine (D) BW25113 M9 Shikimate(A), Uracil (C), 30 <0.1 ≧3 9 Citrate (F) BW25113 M9 Methionine (B),Putrescine (C), 35 <0.1 ≧3 27 Lysine (D) BW25113 M9 Methionine (B),Putrescine (C), 35 <0.1 ≧3 27 Uracil (E) BW25113 M9 Methionine (B),Putrescine (C), 35 <0.1 ≧3 27 Citrate (F) BW25113 M9 Methionine (B),Spermidine (C), 45 <0.1 ≧3 64 Lysine (D) BW25113 M9 Methionine (B),Spermidine (C), 35 <0.1 ≧3 27 Uracil (E) BW25113 M9 Methionine (B),Spermidine (C), 40 <0.1 ≧3 45 Citrate (F) BW25113 M9 Methionine (B),Bicarbonate (C), 45 <0.1 ≧3 64 Lysine (D) BW25113 M9 Methionine (B),Bicarbonate (C), 45 <0.1 ≧3 64 Uracil (E) BW25113 M9 Methionine (B),Bicarbonate (C), 45 <0.1 ≧3 64 Citrate (F) BW25113 M9 Methionine (B),Lysine (D), 35 <0.1 ≧3 27 Uracil (E) BW25113 M9 Homocysteine (B),Bicarbonate (C), 50 <0.1 ≧3 82 Lysine (D) BW25113 M9 Homocysteine (B),Bicarbonate (C), 40 <0.1 ≧3 45 Uracil (E) BW25113 M9 Isoleucine (B),Putrescine (C), 35 <0.1 ≧3 27 Lysine (D) BW25113 M9 Isoleucine (B),Putrescine (C), 30 <0.1 ≧3 9 Uracil (E) BW25113 M9 Isoleucine (B),Putrescine (C), 35 <0.1 ≧3 27 Citrate (F) BW25113 M9 Isoleucine (B),Bicarbonate (C), 55 <0.1 ≧3 100 Lysine (D) BW25113 M9 Isoleucine (B),Bicarbonate (C), 40 <0.1 ≧3 45 Uracil (E) BW25113 M9 Isoleucine (B),Bicarbonate (C), 35 <0.1 ≧3 27 Citrate (F) BW25113 M9 Lysine (B),Bicarbonate (C), 35 <0.1 ≧3 27 Uracil (E) BW25113 M9 Lysine (B),Bicarbonate (C), 35 <0.1 ≧3 27 Citrate (F) BW25113 M9 Methionine (B),Putrescine (C), 30 <0.1 ≧3 9 Lysine (D) BW25113 M9 Methionine (B),Bicarbonate (C), 30 <0.1 ≧3 9 Lysine (D) 4 Supplements BW25113 M9Tyrosine (A), Methionine (B), 50 <0.1 ≧3 82 Putrescine (C), Lysine (D)BW25113 M9 Tyrosine (A), Methionine (B), 40 <0.1 ≧3 45 Putrescine (C),Uracil (E) BW25113 M9 Tyrosine (A), Methionine (B), 35 <0.1 ≧3 27Putrescine (C), Citrate (F) BW25113 M9 Tyrosine (A), Methionine (B), 40<0.1 ≧3 45 Bicarbonate (C), Lysine (D) BW25113 M9 Tyrosine (A),Methionine (B), 40 <0.1 ≧3 45 Bicarbonate (C), Uracil (E) BW25113 M9Tyrosine (A), Methionine (B), 45 <0.1 ≧3 64 Bicarbonate (C), Citrate (F)BW25113 M9 Tyrosine (A), Putrescine (C), 40 <0.1 ≧3 45 Homocysteine (B),Lysine (D) BW25113 M9 Tyrosine (A), Putrescine (C), 30 <0.1 ≧3 9Homocysteine (B), Uracil (E) BW25113 M9 Tyrosine (A), Putrescine (C), 35<0.1 ≧3 27 Homocysteine (B), Citrate (F) BW25113 M9 Tyrosine (A),Bicarbonate (C), 30 <0.1 ≧3 9 Homocysteine (B), Uracil (E) BW25113 M9Tyrosine (A), Bicarbonate (C), 35 <0.1 ≧3 27 Homocysteine (B), Citrate(F) BW25113 M9 Shikimate (A), Putrescine (C), 30 <0.1 ≧3 9 Methionine(B), Lysine (D) BW25113 M9 Shikimate (A), Putrescine (C), 35 <0.1 ≧3 27Methionine (B), Uracil (E) BW25113 M9 Shikimate (A), Putrescine (C), 30<0.1 ≧3 9 Methionine (B), Citrate (F) BW25113 M9 Shikimate (A), Uracil(E), 35 <0.1 ≧3 27 Methionine (B), Lysine (D) BW25113 M9 Shikimate (A),Uracil (E), 35 <0.1 ≧3 27 Methionine (B), Bicarbonate (C) BW25113 M9Shikimate (A), Uracil (E), 30 <0.1 ≧3 9 Methionine (B), Citrate (F)BW25113 M9 Methionine (B), Putrescine (C), 30 <0.1 ≧3 9 Lysine (D),Uracil (E) BW25113 M9 Methionine (B), Bicarbonate (C), 30 <0.1 ≧3 9Lysine (D), Uracil (E) BW25113 M9 Methionine (B), Bicarbonate (C), 35<0.1 ≧3 27 Lysine (D), Citrate (F) BW25113 M9 Bicarbonate (C), Lysine(D), 30 <0.1 ≧3 9 Uracil (E), Citrate (F) BW25113 M9 Methionine (B),Lysine (D), 35 <0.1 ≧3 27 Uracil (E), Citrate (F) 5 supplements BW25113M9 Shikimate (A), Methionine (B), 40 <0.1 ≧3 45 Bicarbonate (C), Lysine(D), Uracil (E) BW25113 M9 Shikimate (A), Homocsyteine (B), 40 <0.1 ≧345 Bicarbonate (C), Lysine (D), Uracil (E) BW25113 M9 Tyrosine (A),Methionine (B), 40 <0.1 ≧3 45 Bicarbonate (C), Lysine (D), Citrate (F)BW25113 M9 Shikimate (A), Methionine (B), 40 <0.1 ≧3 45 Bicarbonate (C),Lysine (D), Citrate (F) BW25113 M9 Shikimate (A), Homocsyteine (B), 40<0.1 ≧3 45 Bicarbonate (C), Lysine (D), Citrate (F) BW25113 M9Methionine (B), Bicarbonate (C), 40 <0.1 ≧3 45 Lysine (D), Uracil (E),Citric (F) BW25113 M9 Tyrosine (A), Methionine (B), 37 <0.1 ≧3 33Bicarbonate (C), Lysine (D), Uracil (E) BW25113 M9 Tyrosine (A),Methionine (B), 35 <0.1 ≧3 27 Putrescine (C), Lysine (D), Uracil (E)BW25113 M9 Shikimate (A), Methionine (B), 35 <0.1 ≧3 27 Putrescine (C),Lysine (D), Uracil (E) BW25113 M9 Tyrosine (A), Homocysteine (B), 35<0.1 ≧3 27 Putrescine (C), Lysine (D), Uracil (E) BW25113 M9 Shikimate(A), Homocsyteine (B), 35 <0.1 ≧3 27 Putrescine (C), Lysine (D), Uracil(E) BW25113 M9 Tyrosine (A), Methionine (B), 35 <0.1 ≧3 27 Putrescine(C), Lysine (D), Citrate (F) BW25113 M9 Tyrosine (A), Homocysteine (B),35 <0.1 ≧3 27 Putrescine (C), Lysine (D), Citrate (F) BW25113 M9Shikimate (A), Homocsyteine (B), 35 <0.1 ≧3 27 Putrescine (C), Lysine(D), Citrate (F) BW25113 M9 Tyrosine (A), Homocysteine (B), 35 <0.1 ≧327 Bicarbonate (C), Lysine (D), Citrate (F) BW25113 M9 Methionine (B),Spermidine (C), 35 <0.1 ≧3 27 Lysine (D), Uracil (E), Citric (F) BW25113M9 Methionine (B), Putrescine (C), 35 <0.1 ≧3 27 Lysine (D), Uracil (E),Citric (F) BW25113 M9 Tyrosine (A), Bicarbonate (C), 35 <0.1 ≧3 27Lysine (D), Uracil (E), Citrate (F) BW25113 M9 Tyrosine (A), Methionine(B), 35 <0.1 ≧3 27 Lysine (D), Uracil (E), Citrate (F) BW25113 M9Shikimate (A), Methionine (B), 35 <0.1 ≧3 27 Lysine (D), Uracil (E),Citrate (F) BW25113 M9 Shikimate (A), Putrescine (C), 30 <0.1 ≧3 9Lysine (D), Uracil (E), Citrate (F) BW25113 M9 Tyrosine (A),Homocysteine (B), 38 <0.1 ≧3 39 Bicarbonate (C), Lysine (D), Uracil (E)BW25113 M9 Shikimate (A), Methionine (B), 30 <0.1 ≧3 9 Putrescine (C),Lysine (D), Citrate (F) 6 supplements BW25113 M9 Tyrosine (A),Methionine (B), 42 <0.1 ≧3 52 Putrescine (C), Lysine (D), Uracil (E),Citrate (F) BW25113 M9 Shikimate (A), Methionine (B), 40 <0.1 ≧3 45Bicarbonate (C), Lysine (D), Uracil (E), Citrate (F) BW25113 M9Shikimate (A), Methionine (B), 35 <0.1 ≧3 27 Putrescine (C), Lysine (D),Uracil (E), Citrate (F) BW25113 M9 Tyrosine (A), Methionine (B), 37 <0.1≧3 33 Bicarbonate (C), Lysine (D), Uracil (E), Citrate (F)

TABLE 48 E. coli Supplement Results under Anaerobic Conditions MIC AssayMIC % Increase Strain Result Assay Over Name Media Supplements (Group)(g/L 3-HP) P-value Number Control CONTROLS BW25113 M9 none 30.0 <0.1 ≧3— BW25113 EZ Rich none 75.0 <0.1 ≧3 150 Single Supplements BW25113 M9Phenylalanine (A) 32.1 <0.1 ≧3 7 BW25113 M9 p-aminobenzoate (A) 40.0<0.1 ≧3 33 BW25113 M9 Dihydroxybenzoate (A) 40.0 <0.1 ≧3 33 BW25113 M9Tetrahydrofolate (A) 40.0 <0.1 ≧3 33 BW25113 M9 Serine (B) 32.1 <0.1 ≧37 BW25113 M9 Methionine (B) 42.8 <0.1 ≧3 43 BW25113 M9 Homoserine (B)30.0 <0.1 ≧3 0 BW25113 M9 Homocysteine Group Mix (B) 45.0 <0.1 ≧3 50BW25113 M9 Putrescine(C) 35.0 <0.1 ≧3 17 BW25113 M9 Spermidine (C) 35.0<0.1 ≧3 17 BW25113 M9 Polyamine Group Mix (C) 60.0 <0.1 ≧3 100 BW25113M9 Lysine (D) 41.7 <0.1 ≧3 39 Double Supplements BW25113 M9Tetrahydrofolate (A), Putrescine (C) 35.0 <0.1 ≧3 17 BW25113 M9Tetrahydrofolate (A), Spermidine (C) 30.0 <0.1 ≧3 0 BW25113 M9Tetrahydrofolate (A), Bicarbonate (C) 35.0 <0.1 ≧3 17 BW25113 M9Tetrahydrofolate (A), Lysine (D) 35.0 <0.1 ≧3 17 BW25113 M9 Homocysteine(B), Bicarbonate (C) 35.0 <0.1 ≧3 17 BW25113 M9 Putrescine (C), Lysine(D) 30.0 <0.1 ≧3 0 BW25113 M9 Putrescine (C), Citrate (F) 36.7 <0.1 ≧322 Triple Supplements BW25113 M9 Methionine (B), Spermidine (C), 35.0<0.1 ≧3 17 Lysine (D) BW25113 M9 Isolucine (B), Putrescine (C), Lysine35.0 <0.1 ≧3 17 (D) 4 Supplements <0.1 ≧3 BW25113 M9 Tyrosine (A),Methionine (B), 40.0 <0.1 ≧3 33 Putrescine (C), Lysine (D) BW25113 M9Tyrosine (A), Methionine (B), 35.0 <0.1 ≧3 17 Bicarbonate (C), Lysine(D) BW25113 M9 Tyrosine (A), Methionine (B), 35.0 <0.1 ≧3 17 Bicarbonate(C), Citrate (F)

Example 50 Evaluation of 3-HPTGC-Related Genetically Modified E. coli

Example 50 provides a direct comparison of one genetic modification ofthe 3HPTC with a control using a growth rate-based toleragram over a24-hour period.

The effects of genetic modifications on 3HP tolerance were determined byMIC evaluations using the methods described in the Common MethodsSection. Genetic modifications tested in E. coli and the MIC resultsthereof are listed in Table 44 for aerobic condition and Table 45 foranaerobic condition. This data, which includes single and multiplegenetic modifications, demonstrates improvement in 3-HP tolerance inthese culture systems based on 24-hour MIC evaluations.

Example 51 Toleragram Comparison with CynTS Genetic Modification

Twenty-four hour duration toleragram evaluations were conducted tocompare a control (wild-type) E. coli (strain BW25113) with agenetically modified E. coli (strain BW25113) comprising a geneticmodification to introduce cynTS.

Results are provided in the figures, which show the control strain alsotested under indicated additional conditions.

Based on the area under the curve, the cynTS treatment is demonstratedto exhibit greater tolerance to 3-HP, at various elevated 3-HPconcentrations, versus the control.

Example 52 Genetic Modification/Introduction of Tolerance Pieces intoBacillus Subtilis

For creation of a 3-HP production tolerance pieces into Bacillussubtilis several genes from the E. coli toleragenic complex were clonedinto a Bacillus shuttle vector, pWH1520 (SEQ ID NO:010) obtained fromBoca Scientific (Boca Raton, Fla. USA). This shuttle vector carries aninducible Pxyl xylose inducible promoter, as well as an ampicillinresistance cassette for propagation in E. coli and a tetracyclineresistance cassette for propagation in Bacillus subtilis. Cloningstrategies for these genes are shown in Table 49.

TABLE 49 B. subtilis Tolerance Plasmid Construction PCR Sequence orCodon Gene(s) Optimized or Region Cloning Sequence Plasmid Name VectorMethod Primer A Primer B (Region) Name speB pWH1520 A SEQID. 0142 SEQID.0143 SEQID. 0144 pWH1520- Pxyl:speB metE pWH1520 A SEQID 0145 SEQID 0146SEQID 0147 pWH1520- Pxyl:metE

Method A

Tolerance genes cloned for testing, in B. subtilis designated a cloningmethod A in Table 49 were created in a similar manner. The cloningmethod described here places the gene under the xylose-induciblepromoter. Each gene was amplified by polymerase chain reaction usingtheir corresponding Primers A and Primer B listed in each row of thetable. Primer A of each set contains homology to the start of the geneand a SpeI restriction site. Primer B contains homology for the regiondownstream of the stop codon of the gene and a BamHI restriction site.The polymerase chain reaction product was purified using a PCRpurification kit obtained from Qiagen Corporation (Valencia, Calif. USA)according to manufacturer's instructions. Next, the purified product wasdigested with SpeI and BamHI obtained from New England BioLabs (Ipswich,Mass. USA) according to manufacturer's instructions. The digestionmixture was separated by agarose gel electrophoresis, and visualizedunder UV transillumination as described in Subsection II of the CommonMethods Section. An agarose gel slice containing a DNA piececorresponding to the digested and purified tolerance gene was cut fromthe gel and the DNA recovered with a standard gel extraction protocoland components from Qiagen (Valencia, Calif. USA) according tomanufacturer's instructions.

This pWH1520 shuttle vector DNA was isolated using a standard miniprepDNA purification kit from Qiagen (Valencia, Calif. USA) according tomanufacturer's instructions. The resulting DNA was restriction digestedwith SpeI and SphI obtained from New England BioLabs (Ipswich, Mass.USA) according to manufacturer's instructions. The digestion mixture wasseparated by agarose gel electrophoresis, and visualized undertransillumination as described in Subsection II of the Common MethodsSection. An agarose gel slice containing a DNA piece corresponding todigested pWH1520 backbone product was cut from the gel and the DNArecovered with a standard gel extraction protocol and components fromQiagen (Valencia, Calif. USA) according to manufacturer's instructions.

Both the digested and purified tolerance gene and pWH1520 DNA productswere ligated together using T4 ligase obtained from New England BioLabs(Ipswich, Mass. USA) according to manufacturer's instructions. Theligation mixture was then transformed into chemically competent 10G E.coli cells obtained from Lucigen Corporation (Middleton Wis., USA)according to the manufacturer's instructions and plated LB platesaugmented with ampicillin for selection. Several of the resultingcolonies were cultured and their DNA was isolated using a standardminiprep DNA purification kit from Qiagen (Valencia, Calif. USA)according to manufacturer's instructions. The recovered DNA was checkedby restriction digest followed by agarose gel electrophoresis. DNAsamples showing the correct banding pattern were further verified by DNAsequencing.

Example 53 Genetic Modification/Introduction of Malonyl-CoA Reductasefor 3HP Production in Bacillus Subtilis

For creation of a 3-HP production pathway in Bacillus Subtilis the codonoptimized nucleotide sequence for the malonyl-coA reductase gene fromChloroflexus aurantiacus that was constructed by the gene synthesisservice from DNA 2.0 (Menlo Park, Calif. USA), a commercial DNA genesynthesis provider, was added to a Bacillus Subtilis shuttle vector.This shuttle vector, pHT08 (SEQ ID NO:011), was obtained from BocaScientific (Boca Raton, Fla. USA) and carries an inducible PgracIPTG-inducible promoter.

This mcr gene sequence was prepared for insertion into the pHT08 shuttlevector by polymerase chain reaction amplification with primer 1(5′GGAAGGATCCATGTCCGGTACGGGTCG-3′) (SEQ ID NO:148), which containshomology to the start site of the mcr gene and a BamHI restriction site,and primer 2 (5′-Phos-GGGATTAGACGGTAATCGCACGACCG-3′) (SEQ ID NO:149),which contains the stop codon of the mcr gene and a phosphorylated 5′terminus for blunt ligation cloning. The polymerase chain reactionproduct was purified using a PCR purification kit obtained from QiagenCorporation (Valencia, Calif. USA) according to manufacturer'sinstructions. Next, the purified product was digested with BamHIobtained from New England BioLabs (Ipswich, Mass. USA) according tomanufacturer's instructions. The digestion mixture was separated byagarose gel electrophoresis, and visualized under UV transilluminationas described in Subsection II of the Common Methods Section. An agarosegel slice containing a DNA piece corresponding to the mcr gene was cutfrom the gel and the DNA recovered with a standard gel extractionprotocol and components from Qiagen (Valencia, Calif. USA) according tomanufacturer's instructions.

This pHT08 shuttle vector DNA was isolated using a standard miniprep DNApurification kit from Qiagen (Valencia, Calif. USA) according tomanufacturer's instructions. The resulting DNA was restriction digestedwith BamHI and SmaI obtained from New England BioLabs (Ipswich, Mass.USA) according to manufacturer's instructions. The digestion mixture wasseparated by agarose gel electrophoresis, and visualized under UVtransillumination as described in Subsection II of the Common MethodsSection. An agarose gel slice containing a DNA piece corresponding todigested pHT08 backbone product was cut from the gel and the DNArecovered with a standard gel extraction protocol and components fromQiagen (Valencia, Calif. USA) according to manufacturer's instructions.

Both the digested and purified mcr and pHT08 products were Ligatedtogether using T4 ligase obtained from New England BioLabs (Ipswich,Mass. USA) according to manufacturer's instructions. The ligationmixture was then transformed into chemically competent 10G E. coli cellsobtained from Lucigen Corporation (Middleton Wis., USA) according to themanufacturer's instructions and plated LB plates augmented withampicillin for selection. Several of the resulting colonies werecultured and their DNA was isolated using a standard miniprep DNApurification kit from Qiagen (Valencia, Calif. USA) according tomanufacturer's instructions. The recovered DNA was checked byrestriction digest followed by agarose gel electrophoresis. DNA samplesshowing the correct banding pattern were further verified by DNAsequencing. The sequence verified DNA was designated as pHT08-mcr, andwas then transformed into chemically competent Bacillus subtilis cellsusing directions obtained from Boca Scientific (Boca Raton, Fla. USA).Bacillus subtilis cells carrying the pHT08-mcr plasmid were selected foron LB plates augmented with chloramphenicol.

Bacillus subtilis cells carrying the pHT08-mcr, were grown overnight in5 ml of LB media supplemented with 20 ug/mL chloramphenicol, shaking at225 rpm and incubated at 37 degrees Celsius. These cultures were used toinoculate 1% v/v, 75 mL of M9 minimal media supplemented with 1.47 g/Lglutamate, 0.021 g/L tryptophan, 20 ug/mL chloramphenicol and 1 mM IPTG.These cultures were then grown for 18 hours in a 250 mL baffledErlenmeyer flask at 25 rpm, incubated at 37 degrees Celsius. After 18hours, cells were pelleted and supernatants subjected to GCMS detectionof 3-HP (described in Common Methods Section IIIb)). Trace amounts of3-HP were detected with qualifier ions.

Example 54 Bacillus Subtilis Strain Construction

Plasmids for tolerance genetic elements in PWH1520 and the productionplasmid, pHT08-mcr, were transformed in to two Bacillus subtilisstrains. The Bacillus subtilis subspecies subtilis 168 strain wasobtained as a kind a gift from the laboratory of Prof. Ryan T. Gill fromthe University of Colorado at Boulder. Transformations were performedusing a modified protocol developed from Anagnostopoulos and Spizizen(Requirements for transformation in Bacillus subtilis. J. Bacteriol.81:741-746 (1961)) as provided with the instructions for the pHT08shuttle vector by Boca Scientific (Boca Raton, Fla. USA).

Example 55 Evaluation of 3HPTGC-Related Supplements on Wild-Type B.Subtilis

The effect of supplementation on 3HP tolerance was determined by MICevaluations using the methods described in the Common Methods Section.Supplements tested are listed in the Supplements Table. Results of theMIC evaluations under anaerobic condition are provided in Table 50.

TABLE 50 B. subtilis Supplement and Genetic Modification Results underAerobic Conditions Chromosomal Vector Based % Increase Group GeneticGenetic Avg 24 hr Standard Over Strain Name Media SupplementsRepresented Parent Modifications Modifications ΔOD600 Error Control B.subtilis 168 M9 + glu + none none NA none none 0.04 0.004 0 trp* B.subtilis 168 M9 + glu + Chorismate A NA none none 0.26 0.043 577 trpGroup B. subtilis 168 M9 + glu + Homocysteine B NA none none 0.08 0.005104 trp Group Mix B. subtilis 168 M9 + glu + Methionine B NA none none0.15 0.007 282 trp B. subtilis 168 M9 + glu + Bicarbonate C NA none none0.06 0.002 56 trp B. subtilis 168 M9 + glu + p- A NA none none 0.070.015 89 trp aminobenzoate B. subtilis 168 M9 + glu + spermidine C NAnone none 0.09 0.024 140 trp B. subtilis 168 M9 + glu + Isoleucine, B,C, D NA none none 0.05 0.006 29 trp Bicarbonate, Lysine B. subtilis 168M9 + glu + Citrate F NA none none 0.30 0.046 674 trp BSX_0003.0 M9 +glu + none none B. subtilis none pWH1520 0.00 0.000 0 trp + 1 mM 168Xylose BSX_0011.0 M9 + glu + none C B. subtilis none pWH1520-Pxyl: 0.070.060 ** trp + 1 mM 168 speB region Xylose BSX_0015.0 M9 + glu + none BB. subtilis none pWH1520-Pxyl: 0.06 0.063 ** trp + 1 mM 168 metE regionXylose *M9 + glu + trp means M9 minimal + glutamate (1.47 g/L) andtryptophan (0.021 g/L) ** Genetically modified strains had a positivechange in growth after 24 hours, compared to control BSX_0003.0 whichhad a decrease in OD600 after 34 hours resulting in a reading of 0.

Example 56 Evaluation of 3HPTGC-Related Genetically Modified B. SubtilisWithout and With 3HPTGC-Related Supplements

The effect of supplementation and/or genetic modifications on 3HPtolerance in B. subtilis was determined by MIC evaluations using themethods described in the Common Methods Section. Supplements tested arelisted in the Supplements Table. Genetic modifications tested and theMIC results under aerobic condition for B. subtilis are provided inTable 50. This data, which includes single genetic modifications andsingle and multiple supplement additions, demonstrates improvement in3-HP tolerance in this culture system based changes in OD.

Example 57 Yeast Aerobic Pathway for 3HP Production (Prophetic)

The following construct (SEQ ID NO:150) containing: 200 bp 5′ homologyto ACC1, His3 gene for selection, Adh1 yeast promoter, BamHI and SpeIsites for cloning of MCR, cycl terminator, Tef1 promoter from yeast andthe first 200 bp of homology to the yeast ACC1 open reading frame willbe constructed using gene synthesis (DNA 2.0). The MCR open readingframe (SEQ ID NO:151) will be cloned into the BamHI and SpeI sites, thiswill allow for constitutive transcription by the adh1 promoter.Following the cloning of MCR into the construct the genetic element (SEQID NO:152) will be isolated from the plasmid by restriction digestionand transformed into relevant yeast strains. The genetic element willknock out the native promoter of yeast ACC1 and replace it with MCRexpressed from the adh1 promoter and the Tef1 promoter will now driveyeast ACC1 expression. The integration will be selected for by growth inthe absence of histidine. Positive colonies will be confirmed by PCR.Expression of MCR and increased expression of ACC1 will be confirmed byRT-PCR.

An alternative approach that could be utilized to express MCR in yeastis expression of MCR from a plasmid. The genetic element containing MCRunder the control of the ADH1 promoter (SEQ ID 4) could be cloned into ayeast vector such as pRS421 (SEQ NO:153) using standard molecularbiology techniques creating a plasmid containing MCR (SEQ ID NO:154). Aplasmid based MCR could then be transformed into different yeaststrains.

Based on the present disclosure, it is noted that, in addition tointroducing a nucleic acid construct that comprises a sequence encodingfor malonyl-CoA reductase activity in a yeast cell, in some embodimentsadditional genetic modifications are made to decrease enoyl-CoAreductase activity and/or other fatty acid synthase activity.

Example 58 Cloning of Saccharomyces Cerevisiae Genetic Elements forIncreased Tolerance to 3HP

Yeast genes were identified by homology and pathway comparison using<<biocyc.org>>, outlined in FIGS. 9W-9BB. Genetic elements wereamplified by PCR using the primers in Table 51. Yeast genetic elementswere amplified to contain native promoters and 3′ untranslated region,PCR product sequences Table 51. PCR products were isolated by gelelectrophoresis and gel purification using Qiagen gel extraction(Valencia, Calif. USA, Cat. No. 28706) following the manufacturesinstructions. Gel purified yeast genetic elements were then cloned intopYes2.1-topo vector (SEQ ID NO:183, Invitrogen Corp, Carlsbad, Calif.,USA) following manufacture instructions. Colonies were screened by PCRand then sequenced by Genewiz.

TABLE 51 Yeast Tolerance Primers Gene Primer A Primer B spe3 SEQID 0155SEQID 0156 hom2 SEQID 0157 SEQID 0158 MET6 SEQID 0159 SEQID 0160 ILV2SEQID 0161 SEQID 0162 ILV6 SEQID 0163 SEQID 0164 THR1 SEQID 0165 SEQID0166 SER2 SEQID 0167 SEQID 0168 SER3 SEQID 0169 SEQID 0170 ARG2 SEQID0171 SEQID 0172 RNR1 SEQID 0173 SEQID 0174 aro3 SEQID 0175 SEQID 0176ARO7 SEQID 0177 SEQID 0178 TYR1 SEQID 0179 SEQID 0180 TRP1 SEQID 0181SEQID 0182

Example 59 Sub-Cloning Yeast Genetic Elements into E. coli/Yeast ShuttleVectors pRS423 and pRS425

Genetic elements were excised from pYes2.1 by restriction digestion withrestriction enzymes PvulI and XbaI. Restriction fragments containingyeast genetic elements were isolated by gel electrophoresis and gelpurification using Qiagen gel extraction (Valencia, Calif. USA, Cat. No.28706) following manufactures instructions. Backbone vectors pRS423 andpRS425 were digested with SmaI and SpeI restriction enzymes and gelpurified. Yeast genetic elements were ligated into pRS423 and pRS425(SEQ ID NO:184 and 185). All plasmids were checked using PCR analysisand sequencing.

Example 60 Yeast Strain Construction

Yeast strains were constructed using standard yeast transformation andselected for by complementation of auxotrophic markers. All strains areS288C background. For general yeast transformation methods, see Gietz,R. D. and R. A. Woods. (2002) “Transformation of Yeast by the Liac/SSCarrier DNA/PEG Method.” Methods in Enzymology 350: 87-96.

Example 61 Evaluation of Supplements and/or Genetic Modifications on 3HPTolerance in Yeast

The effect of supplementation and/or genetic modifications on 3HPtolerance was determined by MIC evaluations using the methods describedin this Example. Supplements tested are listed in Tables 52 and 53 foraerobic and anaerobic conditions, respectively. Genetic modificationstested in yeast are listed in Tables 54 and 55 for aerobic and anaerobicconditions, respectively. Results of the MIC evaluations are provided inTables 52-55. This data, which includes single and multiple supplementadditions and genetic modifications, demonstrates improvement in 3-HPtolerance in these culture systems based on the MIC evaluationsdescribed herein.

Method for Yeast Aerobic Minimum Inhibitory Concentration Evaluation

The minimum inhibitory concentration (MIC) was determined aerobically ina 96 well-plate format. Plates were setup such that each individualwell, when brought to a final volume of 100 uL following inoculation,had the following component levels (corresponding to synthetic minimalglucose medium (SD) standard media without vitamins): 20 g/L dextrose, 5g/L ammonium sulfate, 850 mg/L potassium phosphate monobasic, 150 mg/Lpotassium phosphate dibasic, 500 mg/L magnesium sulfate, 100 mg/L sodiumchloride, 100 mg/L calcium chloride, 500 μg/L boric acid, 40 μg/L coppersulfate, 100 μg/L potassium iodide, 200 μg/L ferric chloride, 400 μg/Lmanganese sulfate, 200 μg/L sodium molybdate, and 400 μg/L zinc sulfate.Media supplements were added according to levels reported in theSupplements Table, where specified. Overnight cultures of strains weregrown in triplicate in 5 mL SD media with vitamins (Methods inEnzymology vol. 350, page 17 (2002)). A 1% (v/v) inoculum was introducedinto a 5 ml culture of SD minimal media without vitamins. After thecells reached mid-exponential phase, the culture was diluted to an OD₆₀₀of 0.200. The cells were further diluted 1:5 and a 10 μL aliquot wasused to inoculate each well of a 96 well plate (˜10⁴ cells per well) tototal volume of 100 uL. The plate was arranged to measure the growth ofvariable strains or growth conditions in increasing 3-HP concentrations,0 to 60 g/L, in 5 g/L, increments. Plates were incubated for 72 hours at30 C. The minimum inhibitory 3-HP concentration and maximum 3-HPconcentration corresponding to visible cell growth (OD˜0.1) was recordedafter 72 hours. For cases when MIC >60 g/L, assessments were performedin plates with extended 3-HP concentrations (0-100 g/L, in 5 g/Lincrements).

Method for Yeast Anaerobic Minimum Inhibitory Concentration Evaluation

The minimum inhibitory concentration (MIC) was determined anaerobicallyin a 96 well-plate format. Plates were setup such that each individualwell, when brought to a final volume of 100 uL following inoculation,had the following component levels (corresponding to synthetic minimalglucose medium (SD) standard media without vitamins): 20 g/L dextrose, 5g/L ammonium sulfate, 850 mg/L potassium phosphate monobasic, 1.50 mg/Lpotassium phosphate dibasic, 500 mg/L magnesium sulfate, 100 mg/L sodiumchloride, 100 mg/L calcium chloride, 500 ug/L boric acid, 40 ug/L coppersulfate, 100 ug/L potassium iodide, 200 ug/L, ferric chloride, 400 ug/Lmanganese sulfate, 200 ug/L sodium molybdate, and 400 ug/L zinc sulfate.Overnight cultures of strains were grown in triplicate in 5 mL SD mediawith vitamins (Methods in Enzymology vol. 350, page 17 (2002)). A 1%(v/v) inoculum was introduced into a 5 ml culture of SD minimal mediawithout vitamins. After the cells reached mid-exponential phase, theculture was diluted to an OD₆₀₀ of 0.200. The cells were further diluted1:5 and a 10 μL aliquot was used to inoculate each well of a 96 wellplate (˜10⁴ cells per well) to total volume of 100 uL. The plate wasarranged to measure the growth of variable strains or growth conditionsin increasing 3-HP concentrations, 0 to 60 g/L, in 5 g/L, increments.Plates were incubated for 72 hours at 30 C. The minimum inhibitory 3-HPconcentration and maximum 3-HP concentration corresponding to visiblecell growth (OD˜0.1) was recorded after 72 hours. For cases when MIC >60g/L, assessments were performed in plates with extended 3-HPconcentrations (0-100 g/L, in 5 gL increments). Plates were sealed inbiobag anaerobic chambers that contained gas generators for anaerobicconditions and incubated for 72 hours at 30 C. The minimum inhibitory3-HP concentration and maximum 3-HP concentration corresponding tovisible cell growth (OD˜0.1) was recorded after 72 hours. For cases whenMIC >60 g/L, assessments were performed in plates with extended 3-HPconcentrations (0-100 g/L, in 5 g/L increments).

TABLE 52 Yeast Supplement Results Under Aerobic Conditions Average MICMIC % Increase Strain Assay Result Assay Over Name Media Supplements(Group) (g/L 3-HP) S.D. Number Control CONTROLS S288C SD none 45 2.5 ≧3— S288C SC none 60 <2.5 ≧3 33 S288C SD Tryptophan (A) 54 17.4 ≧3 20S288C SD Shikimate (A) 80 <2.5 ≧3 78 S288C SD Chorismate Group Mix (A)80 <2.5 ≧3 78 S288C SD Glycine (B) 50 11.0 ≧3 11 S288C SD Methionine (B)72 16.9 ≧3 59 S288C SD 2-oxobutyrate (B) 50 <2.5 ≧3 11 S288C SDAspartate 57 2.9 ≧3 26 S288C SD Homocysteine Group Mix (B) 87 5.8 ≧3 93S288C SD Putrescine(C) 55 16.4 ≧3 22 S288C SD Citrulline (C) 58 21.4 ≧328 Supplement Combinations Control S288C SD none 45 2.5 ≧3 — S288C SDTyrosine (A), Methionine (B), Putrescine 77 4.7 ≧3 70 (C), Lysine (D)S288C SD Methionine (B), Ornithine (C) 80 0.0 ≧3 78 S288C SDHomocysteine (B), Spermidine (C) 77 4.7 ≧3 70 S288C SD Tyrosine (A),Bicarbonate (C), Lysine 70 <2.5 ≧3 56 (D) S288C SD Tyrosine (A),Bicarbonate (C), Uracil (E) 67 4.7 ≧3 48 S288C SD Methionine (B),Spermidine (C), Lysine 77 4.7 ≧3 70 (D) S288C SD Methionine (B),Bicarbonate (C), Lysine 70 <2.5 ≧3 56 (D) S288C SD Methionine (B),Bicarbonate (C), Uracil 77 4.7 ≧3 70 (E) S288C SD Methionine (B),Bicarbonate (C), Citrate 50 <2.5 ≧3 11 (F) S288C SD Putrescine (C),Lysine (D) 57 4.7 ≧3 26 S288C SD Tyrosine (A), Methionine (B),Putrescine 77 4.7 ≧3 70 (C), Lysine (D), Uracil (E), Citrate (F) S288CSD Tyrosine (A), Putrescine (C) 77 4.7 ≧3 70 S288C SD Tetrahydrofolate(A), Spermidine (C) 70 <2.5 ≧3 56 S288C SD Homocysteine (B), Putrescine(C) 80 <2.5 ≧3 78 S288C SD Spermidine (C), Lysine (D) 70 <2.5 ≧3 56S288C SD Bicarbonate (C), Citrate (F) 50 <2.5 ≧3 11 S288C SD Tyrosine(A), Bicarbonate (C), Citrate (F) 50 <2.5 ≧3 11 S288C SD Methionine (B),Spermidine (C), Citrate 67 4.7 ≧3 48 (F) S288C SD Homocysteine (B),Bicarbonate (C), 60 <2.5 ≧3 33 Uracil (E)

TABLE 53 Yeast Supplement Results Under Anaerobic Conditions Average MICMIC % Increase Strain Assay Result Assay Over Name Media Supplements(Group) (g/L 3-HP) S.D. Number Control CONTROLS S288C SD none 38 2.7 ≧3— S288C SD Phenylalanine (A) 38 2.9 ≧3 2 S288C SD Tryptophan (A) 55 5.5≧3 47 S288C SD Shikimate (A) 60 <2.5 ≧3 60 S288C SD Chorismate Group Mix(A) 48 4.1 ≧3 29 S288C SD Homocysteine (B) 40 <2.5 ≧3 7 S288C SDIsoleucine (B) 38 2.9 ≧3 2 S288C SD Serine (B) 45 <2.5 ≧3 20 S288C SDGlycine (B) 60 <2.5 ≧3 60 S288C SD Methionine (B) 100 <2.5 ≧3 167 S288CSD Threonine (B) 38 2.9 ≧3 2 S288C SD 2-oxobutyrate (B) 38 2.9 ≧3 2S288C SD Homocysteine Group Mix (B) 100 <2.5 ≧3 167 S288C SDPutrescine(C) 58 4.1 ≧3 56 S288C SD Cadaverine (C) 60 4.1 ≧3 60 S288C SDSpermidine (C) 60 <2.5 ≧3 60 S288C SD Citrulline (C) 97 5.8 ≧3 158 S288CSD Bicarbonate (C) 90 <2.5 ≧3 140 S288C SD Polyamine Group Mix (C) 422.9 ≧3 11 S288C SD Lysine (D) 45 <2.5 ≧3 20 Supplement CombinationsControl S288C SD none 38 2.7 ≧3 0 S288C SD Isoleucine (B), Bicarbonate(C), 67 <2.5 ≧3 78 Lysine (D) S288C SD Homocysteine (B), Bicarbonate 80<2.5 ≧3 113 (C), Lysine (D) S288C SD Tyrosine (A), Methionine (B), 554.7 ≧3 47 Putrescine (C), Lysine (D) S288C SD Methionine (B), Putrescine(C) 55 <2.5 ≧3 47 S288C SD Methionine (B), Ornithine (C) 50 <2.5 ≧3 33S288C SD Homocysteine (B), Spermidine 40 4.7 ≧3 7 (C) S288C SD Tyrosine(A), Bicarbonate (C), 70 <2.5 ≧3 87 Lysine (D) S288C SD Tyrosine (A),Bicarbonate (C), 50 4.7 ≧3 33 Uracil (E) S288C SD Methionine (B),Spermidine (C), 100 4.7 ≧3 167 Lysine (D) S288C SD Methionine (B),Bicarbonate 80 <2.5 ≧3 113 (C), Lysine (D) S288C SD Methionine (B),Bicarbonate 78 4.7 ≧3 107 (C), Uracil (E) S288C SD Methionine (B),Bicarbonate 73 <2.5 ≧3 93 (C), Citrate (F) S288C SD Homocysteine (B),Bicarbonate 77 <2.5 ≧3 104 (C) S288C SD Putrescine (C), Lysine (D) 77<2.5 ≧3 104 S288C SD Tyrosine (A), Methionine (B), 68 4.7 ≧3 82Putrescine (C), Lysine (D), Uracil (E), Citrate (F) S288C SD Tyrosine(A), Putrescine (C) 57 4.7 ≧3 51 S288C SD Tyrosine (A), Spermidine (C)60 4.7 ≧3 60 S288C SD Tetrahydrofolate (A), 50 <2.5 ≧3 33 Spermidine (C)S288C SD Methionine (B), Spermidine (C) 50 <2.5 ≧3 33 S288C SDHomocysteine (B), Putrescine 100 <2.5 ≧3 167 (C) S288C SD Spermidine(C), Lysine (D) 100 <2.5 ≧3 167 S288C SD Bicarbonate (C), Citrate (F) 50<2.5 ≧3 33 S288C SD Tyrosine (A), Methionine (B), 40 <2.5 ≧3 7 Uracil(E) S288C SD Tyrosine (A), Bicarbonate (C), 50 <2.5 ≧3 33 Citrate (F)S288C SD Methionine (B), Spermidine (C), 50 <2.5 ≧3 33 Citrate (F) S288CSD Homocysteine (B), Bicarbonate 57 4.7 ≧3 51 (C), Uracil (E)

TABLE 54 Yeast Genetic Modification Results Under Aerobic ConditionsVector based MIC Assay MIC % Increase Group Genetic Result (g/L AssayOver Strain Name Media Represented Parent Modifications 3-HP) S.D.Number Control YX-CJR-001 SD none BY4709 pRS426 EV 40 <2.5 ≧3 —YX-CJR-002 SD C BY4709 pYes2.1-spe3 50 <2.5 ≧3 25 YX-CJR-003 SD B BY4709pYes2.1-hom2 47 <2.5 ≧3 17 YX-CJR-005 SD B BY4709 pYes2.1-Met6 50 <2.5≧3 25 YX-CJR-006 SD B BY4709 pYes2.1-Ilv2 57 <2.5 ≧3 42 YX-CJR-010 SD BBY4709 pyes2.1-Thr1 60 <2.5 ≧3 50 YX-CJR-014 SD C BY4709 pyes2.1-arg2 60<2.5 ≧3 50 YX-CJR-017 SD A BY4709 pyes2.1-Aro7 70 <2.5 ≧3 75 YX-022 SDA, B BY4722 pyes2.1-Aro3 60 <2.5 ≧3 50 pRS425-ILV6

TABLE 55 Yeast Genetic Modification Results Under Anaerobic ConditionsMIC Assay MIC % Increase Group Vector based Genetic Result Assay OverStrain Name Media Represented Parent Modifications (g/L 3-HP) P-valueNumber Control YX-CJR-001 SD none BY4709 pRS426 EV 40 <0.1 ≧3 —YX-CJR-005 SD B BY4709 pYes2.1-Met6 60 <0.1 ≧3 50 YX-CJR-007 SD B BY4709pyes2.1-ILV6 50 <0.1 ≧3 25 YX-CJR-008 SD B BY4709 pyes2.1-ILV1 60 <0.1≧3 50 YX-CJR-010 SD B BY4709 pyes2.1-Thr1 50 <0.1 ≧3 25 YX-CJR-011 SD BBY4709 pyes2.1-Ser2 50 <0.1 ≧3 25 YX-CJR-013 SD B BY4709 pyes2.1-ser3 50<0.1 ≧3 25 YX-CJR-014 SD C BY4709 pyes2.1-arg2 50 <0.1 ≧3 25 YX-CJR-015SD E BY4709 pyes2.1-RNR1 50 <0.1 ≧3 25 YX-CJR-016 SD A BY4709pyes2.1-Aro3 50 <0.1 ≧3 25 YX-CJR-018 SD A BY4709 pyes2.1-Tyr1 50 <0.1≧3 25 YX-CJR-021 SD A BY4709 pYes2.1-Trp1 50 <0.1 ≧3 25 YX-022 SD A, BBY4722 pyes2.1-Aro3 pRS425- 50 <0.1 ≧3 25 ILV6

TABLE 56 C. necator Supplement Results under Aerobic Conditions averageMIC MIC Strain Supplement Assay Result Assay % Increase Name MediaSupplements Codes (g/L 3-HP) P-value Number Over Control DSM428 FGN nonenone 15 <0.1 ≧3 — DSM 542 EZ Rich none none 60 <0.1 ≧3 200 DSM 542 FGNnone none 15 <0.1 ≧3 0 DSM 542 FGN Homocysteine Bicarbonate, Lysine B,C, D 30 <0.1 ≧3 100 DSM 542 FGN Tyrosine, Methionine, Putrescine, A, B,C, D 30 <0.1 ≧3 100 Lysine DSM 542 FGN Methionine, Putrescine B, C 25<0.1 ≧3 67 DSM 542 FGN Methionine, Ornithine B, C 30 <0.1 ≧3 100 DSM 542FGN Homocysteine, Spermidine B, C 25 <0.1 ≧3 67 DSM 542 FGN Methionine,Bicarbonate, Citrate B, C, F 25 <0.1 ≧3 67 DSM 542 FGN Homocysteine,Bicarbonate B, C 25 <0.1 ≧3 67 DSM 542 FGN Homocysteine Group Mix B 20<0.1 ≧3 33

Example 62 Evaluation of 3-HPTGC-Related Supplements in CupriavidusNecator

The effect of supplementation on 3HP tolerance in C. necator wasdetermined by MIC evaluations using the methods described in the CommonMethods Section. Supplements tested are listed in the Supplements Table.

MIC results under aerobic condition for C. necator are provided in Table56. This data, which includes single and multiple supplement additions,demonstrates improvement in 3-HP tolerance in these culture systemsbased on the MIC evaluations.

Example 63 Additional Example of 3HPTGC Tolerance-Directed GeneticModification(s) in Combination with 3-HP Production GeneticModification(s)

In addition to Example 42, which provides a general example to combinetolerance and 3-HP production genetic modifications to obtain a desiredgenetically modified microorganism suitable for use to produce 3-HP, andin view of the examples following Example 43, and considering additionaldisclosure herein, and methods known to those skilled in the art (e.g.,Sambrook and Russell, 2001, incorporated into this example for itsmethods of genetic modifications), this example provides a microorganismspecies genetically modified to comprise one or more geneticmodifications of the 3HPTGC to provide an increase tolerance to 3-HP(which may be assessed by any metric such as those discussed herein) andone or mare genetic modifications to increase 3-HP production (such asof a 3-HP production pathway such as those disclosed herein).

The so-genetically modified microorganism may be evaluated both fortolerance to and production of 3-HP under varying conditions includingoxygen content of the culture system and nutrient composition of themedia.

In various aspects of this example, multiple sets of geneticmodifications are made and are compared to identify one or moregenetically modified microorganisms that comprise desired attributesand/or metrics for increased 3-HP tolerance and production.

Example 64 Introduction of Genetic Modification Encoding the IrokSequence Combined with 3HPTGC Genetic Modifications

Example 45 describes Irok, a peptide comprised of 21 amino acids, andits 3-HP tolerance improving effect when a plasmid encoding it isintroduced into an E. coli strain and evaluated under microaerohicconditions. Considering the disclosure herein regarding the 3HPTGC, andmethods known to those skilled in the art (e.g., Sambrook and Russell,2001, incorporated into this example for its methods of geneticmodifications), a microorganism species is genetically modified tocomprise a nucleic acid sequence that encodes the IroK peptide sequenceand one or more genetic modifications of the 3-HPTGC, collectively toprovide an increase tolerance to 3-HP. Such increase in 3-HP tolerancemay be assessed by any metric such as those discussed herein.

Thus, based on the results various genetic modification combinationsthat include representation from two or more of the Groups A-E may beevaluated, and employed, in a microorganism to achieve a desiredelevated tolerance to 3-HP. The tables above show the results ofparticular genetic modification combinations that include combinationsfrom these groups. Also, additional genetic modifications may beprovided from Group F. As described elsewhere herein, any suchcombination may be combined with other genetic modifications that mayinclude one or more of: 3-HP bio-production pathways to provide and/orincrease 3-HP synthesis and accumulation by the recombinantmicroorganism, and deletions or other modifications to direct moremetabolic resources (e.g., carbon and energy) into 3-HP bio-production,as well as other genetic modifications directed to modulate flux intothe fatty acid synthase system.

The following are non-limiting general prophetic examples directed topracticing the present invention in other microorganism species.

General Prophetic Example 65 Improvement 3-HP Tolerance and/orBio-Production in Rhodococcus Erythropolis

A series of E. coli-Rhodococcus shuttle vectors are available forexpression in R. erythropolis, including, but not limited to, pRhBR17and pDA71 (Kostichka et al., Appl. Microbiol. Biotechnol.62:61-68(2003)). Additionally, a series of promoters are available forheterologous gene expression in R. erythropolis (see for exampleNakashima et al., Appl. Environ, Microbiol, 70:5557-5568 (2004), and Taoet al,, Appl. Microbiol. Biotechnol. 2005, DOI 10.1007/s00253-005-0064).Targeted gene disruption of chromosomal genes in R. erythropolis may becreated using the method described by Tao et al., supra, and Brans etal. (Appl. Environ. Microbiol. 66: 2029-2036 (2000)). These publishedresources are incorporated by reference for their respective indicatedteachings and compositions.

The nucleic acid sequences required for providing an increase in 3-HPtolerance, as described herein, optionally with nucleic acid sequencesto provide and/or improve a 3-HP biosynthesis pathway, are clonedinitially in pDA71 or pR1aBR71 and transformed into E. coli. The vectorsare then transformed into R. erythropolis by electroporation, asdescribed by Kostichka et al., supra. The recombinants are grown insynthetic medium containing glucose and the tolerance to and/orbio-production of 3-HP are followed using methods known in the art ordescribed herein.

General Prophetic Example 66 Improvement of 3-HP Tolerance and/orBio-Production in B. Licheniformis

Most of the plasmids and shuttle vectors that replicate in B. subtilisare used to transform B. licheniformis by either protoplasttransformation or electroporation. The nucleic acid sequences requiredfor improvement of 3-HP tolerance, and/or for 3-HP biosynthesis areisolated from various sources, codon optimized as appropriate, andcloned in plasmids pBE20 or pBE60 derivatives (Nagarajan et al., Gene114:121-126 (1992)). Methods to transform B. licheniformis are known inthe art (for example see Fleming et al, Appl, Environ. Microbiol.,61(11):3775-3780 (1995)). These published resources are incorporated byreference for their respective indicated teachings and compositions.

The plasmids constructed for expression in B. subtilis are transformedinto B. licheniformis to produce a recombinant microorganism that thendemonstrates improved 3-HP tolerance, and, optionally, 3-HPbio-production.

General Prophetic Example 67 Improvement of 3-HP Tolerance and/orBio-Production in Paenibacillus Macerans

Plasmids are constructed as described herein for expression in B.subtilis and used to transform Paenibacillus macerans by protoplasttransformation to produce a recombinant microorganism that demonstratesimproved 3-HP tolerance, and, optionally, 3-HP bio-production.

General Prophetic Example 68 Expression of 3-HP Tolerance and/orBio-Production in Alcaligenes (Ralstonia) eutraphus (Currently Referredto as Cupriavidus Necator)

Methods for gene expression and creation of mutations in Alcaligeneseutrophus are known in the art (see for example Taghavi et al., Appl.Environ. Microbiol., 60(10);3585-3591 (1994)). This published resourceis incorporated by reference for its indicated teachings andcompositions. Any of the nucleic acid sequences identified to improve3-HP tolerance, and/or for 3-HP biosynthesis are isolated from varioussources, codon optimized as appropriate, and cloned in any of the broadhost range vectors described herein, and electroporated to generaterecombinant microorganisms that demonstrate improved 3-HP tolerance,and, optionally, 3-HP bio-production. The poly(hydroxybutyrate) pathwayin Alcaligenes has been described in detail, a variety of genetictechniques to modify the Alcaligenes eutrophus genome is known, andthose tools can be applied for engineering a 3-HP toleragenic or,optionally, a 3-HP-gena-toleragenic recombinant microorganism.

General Prophetic Example 69 Improvement of 3-HP Tolerance and/orBio-Production in Pseudomonas Putida

Methods for gene expression in Pseudomonas putida are known in the art(see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which isincorporated herein by reference for these teachings). Any of thenucleic acid sequences identified to improve 3-HP tolerance, and/or for3-HP biosynthesis are isolated from various sources, codon optimized asappropriate, and cloned in any of the broad host range vectors describedherein, and electroporated to generate recombinant .microorganisms thatdemonstrate improved 3-HP tolerance, and, optionally, 3-HP biosyntheticproduction. For example, these nucleic acid sequences are inserted intopUCP18 and this ligated DNA are electroporated into electrocompetentPseudomonas putida KT2440 cells to generate recombinant P. putidamicroorganisms that exhibit increased 3-HP tolerance and optionally alsocomprise 3-HP biosynthesis pathways comprised at least in part ofintroduced nucleic acid sequences.

General Prophetic Example 70 Improvement of 3-HP Tolerance and/orBio-Production in Lactobacillus Plantarum

The Lactobacillus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation or Bacillus subtilis andStreptococcus are used for Lactobacillus. Non-limiting examples ofsuitable vectors include pAM.beta.1 and derivatives thereof (Renault etal., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231(1993)); pMG1, and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl.Environ. Microbiol 62:1481-1486 (1996)); pMG1, a conjugative plasmid(Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520(Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997));pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001));and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903(1994)). Several plasmids from Lactobacillus plantarum have also beenreported (e.g., van Kranenburg R, Golic N, Bangers R, Leer R J. de Vos WM, Siezen R J, Kleerebezem M. Appl. Environ. Microbiol. 2005 March;71(3): 1223-1230).

General Prophetic Example 71 Improvement of 3-HP Tolerance and/orBio-Production in Enterococcus faecium, Enterococcus gallinarium, andEnterococcus faecalis

The Enterococcus genus belongs to the Lactobacillales family and manyplasmids and vectors used in the transformation of Lactobacillus,Bacillus subtilis, and Streptococcus are used for Enterococcus.Non-limiting examples of suitable vectors include pAM.beta.1 andderivatives thereof (Renault et al., Gene 183:175-182 (1996); andO'Sullivan et al., Gene 137:227-231 (1993)); pMBB1 and pHW800, aderivative of pMBB1 (Wyckoff et al. Appl. Environ. Microbiol.62;1481-1486 (1996)); pMG1, a conjugative plasmid (Tanimoto et al., J.Bacteriol. 184:5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl.Environ. Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl.Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al.,Antimicrob. Agents Chemother. 38:1899-1903 (1994)). Expression vectorsfor E. faecalis using the nisA gene from Lactococcus may also be used(Eichenbaum et al., Appl. Environ. Microbiol. 64:2763-2769 (1998).Additionally, vectors for gene replacement in the E. faecium chromosomeare used (Nallaapareddy et al., Appl. Environ. Microbiol. 72:334-345(2006)).

For each of the General Prophetic Examples 65-71, the following 3-HPbio-production comparison may be incorporated thereto: Using analyticalmethods for 3-HP such as are described in Subsection III of CommonMethods Section, 3-HP is obtained in a measurable quantity at theconclusion of a respective bio-production event conducted with therespective recombinant microorganism (see types of bio-productionevents, incorporated by reference into each respective General PropheticExample). That measurable quantity is substantially greater than aquantity of 3-HP produced in a control bio-production event using asuitable respective control microorganism lacking the functional 3-HPpathway so provided in the respective General Prophetic Example.Tolerance improvements also may be assessed by any recognizedcomparative measurement technique, such as by using a MIC protocolprovided in the Common Methods Section.

Common Methods Section

All methods in this Section are provided for incorporation into theExamples where so referenced.

Subsection I. Microorganism Species and Strains, Cultures, and Growth

Bacterial species, that may be utilized as needed, are as follows:

Acinetobacter calcoaceticus (DSMZ #1139) is obtained from the GermanCollection of Microorganisms and Cell Cultures (Braunschweig, Germany)as a vacuum dried culture. Cultures are then resuspended in Brain HeartInfusion (BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serialdilutions of the resuspended A. calcoaceticus culture are made into BHIand are allowed to grow for aerobically for 48 hours at 37° C. at 250rpm until saturated.

Bacillus subtilis is a gift from the Gill lab (University of Colorado atBoulder) and is obtained as an actively growing culture. Serialdilutions of the actively growing B. subtilis culture are made intoLuria Broth (RPI Corp, Mt. Prospect, Ill., USA) and are allowed to growfor aerobically for 24 hours at 37° C. at 250 rpm until saturated.

Chlorobium limicola (DSMZ#245) is obtained from the German Collection ofMicroorganisms and Cell Cultures (Braunschweig, Germany) as a vacuumdried culture. Cultures are then resuspended using Pfennig's Medium Iand II (#28 and 29) as described per DSMZ instructions. C. limicola isgrown at 25° C. under constant vortexing.

Citrobacter braakii (DSMZ #30040) is obtained from the German Collectionof Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuumdried culture. Cultures are then resuspended in Brain Heart Infusion(BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serial dilutions of theresuspended C. braakii culture are made into BHI and are allowed to growfor aerobically for 48 hours at 30° C. at 250 rpm until saturated.

Clostridium acetobutylicum (DSMZ #792) is obtained from the GermanCollection of Microorganisms and Cell Cultures (Braunschweig, Germany)as a vacuum dried culture. Cultures are then resuspended in Clostridiumacetobutylicum medium (#411) as described per DSMZ instructions. C.acetobutylicum is grown anaerobically at 37° C. at 250 rpm untilsaturated.

Clostridium aminobutyricum (DSMZ #2634) is obtained from the GermanCollection of Microorganisms and Cell Cultures (Braunschweig, Germany)as a vacuum dried culture. Cultures are then resuspended in Clostridiumaminobutyricum medium (#286) as described per DSMZ instructions. C.aminobutyricum is grown anaerohically at 37° C. at 250 rpm untilsaturated.

Clostridium kluyveri (DSMZ #555) is obtained from the German Collectionof Microorganisms and Cell Cultures (Braunschweig, Germany) as anactively growing culture. Serial dilutions of C. kluyveri culture aremade into Clostridium kluyveri medium (#286) as described per DSMZinstructions. C. kluyveri is grown anaerobically at 37° C. at 250 rpmuntil saturated.

Cupriaridus metallidurans (DMSZ #2839) is obtained from the GermanCollection of Microorganisms and Cell Cultures (Braunschweig, Germany)as a vacuum dried culture. Cultures are then resuspended in Brain HeartInfusion (BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serialdilutions of the resuspended C. metallidurans culture are made into BHIand are allowed to grow for aerobically for 48 hours at 30° C. at 250rpm until saturated.

Cupriavidus necator (DSMZ 428) is obtained from the German Collection ofMicroorganisms and Cell Cultures (Braunschweig, Germany) as a vacuumdried culture. Cultures are then resuspended in Brain Heart Infusion(BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serial dilutions of theresuspended C. necator culture are made into BHI and are allowed to growfor aerobically for 48 hours at 30° C. at 250 rpm until saturated. Asnoted elsewhere, previous names for this species are Alcaligeneseutrophus and Ralstonia eutrophus.

Desulfovibrio fructosovorans (DSMZ #3604) is obtained from the GermanCollection of Microorganisms and Cell Cultures (Braunschweig, Germany)as a vacuum dried culture. Cultures are then resuspended inDesulfovibrio fructosovorans medium (#63) as described per DSMZinstructions. D. fructosovorans is grown anaerobically at 37° C. at 250rpm until saturated.

Escherichia coli Crooks (DSMZ#1576) is obtained from the GermanCollection of Microorganisms and Cell Cultures (Braunschweig, Germany)as a vacuum dried culture. Cultures are then resuspended in Brain HeartInfusion (BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serialdilutions of the resuspended E. coli Crooks culture are made into BHIand are allowed to grow for aerobically for 48 hours at 37° C. at 250rpm until saturated.

Escherichia coli K12 is a gift from the Gill lab (University of Coloradoat Boulder) and is obtained as an actively growing culture. Serialdilutions of the actively growing E. coli K12 culture are made intoLuria Broth (RPI Corp, Mt. Prospect, Ill., USA) and are allowed to growfor aerobically for 24 hours at 37° C. at 250 rpm until saturated.

Halobacterium salinarum (DSMZ#1576) is obtained from the GermanCollection of Microorganisms and Cell Cultures (Braunschweig, Germany)as a vacuum dried culture. Cultures are then resuspended inHalobacterium medium (#97) as described per DSMZ instructions. H.salinarum is grown aerobically at 37° C. at 250 rpm until saturated.

Lactobacillus delbrueckii (#4335) is obtained from WYEAST USA (Odell,Oreg., USA) as an actively growing culture. Serial dilutions of theactively growing L. delbrueckii culture are made into Brain HeartInfusion (BHI) broth (RPI Corp, Mt. Prospect, Ill., USA) and are allowedto grow for aerobically for 24 hours at 30° C. at 250 rpm untilsaturated.

Metallosphaera sedula (DSMZ #5348) is obtained from the GermanCrillection of Microorganisms and Cell Cultures (Braunschweig, Germany)as an actively growing culture. Serial dilutions of M. sedula cultureare made into Metallosphaera medium (#485) as described per DSMZinstructions. M. sedula is grown aerobically at 65° C. at 250 rpm untilsaturated.

Propionibacterium freudenreichii subsp. shermanii (DSMZ#4902) isobtained from the German Collection of Microorganisms and Cell Cultures(Braunschweig, Germany) as a vacuum dried culture. Cultures are thenresuspended in PYG-medium (#104) as described per DSMZ instructions. P.freudenreichii subsp. shermanii is grown anaerobicallly at 30° C. at 250rpm until saturated.

Pseudomonas putida is a gift from the Gill lab (University of Coloradoat Boulder) and is obtained as an actively growing culture. Serialdilutions of the actively growing P. putida culture are made into LuriaBroth (RPI Corp, Mt. Prospect, Ill., USA) and are allowed to grow foraerobically for 24 hours at 37° C. at 250 rpm until saturated.

Streptococcus mutans (DSMZ#6178) is obtained from the German Collectionof Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuumdried culture. Cultures are then resuspended in Luria Broth (RPI Corp,Mt. Prospect, Ill., USA). S. nutans is grown aerobically at 37° C. at250 rpm until saturated.

The following non-limiting strains may also he used as starting strainsin the Examples:DF40 Hfr(PO2A), garB10, fhuA22, ompF627(T2R),fadL701(T2R), relA1, pitA10, spoT1, rrnB-2, pgi-2, mcrB1, creC510,BW25113 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), &lambda⁻, rph-1,Δ(rhaD-rhaB)568, hsdR514, JP111 Hfr(PO1), galE45(GalS), &lambda⁻,fab1392(ts), relA1, spoT1, thi-1. These strains possess recognizedgenetic modifications, and are available from public culture sourcessuch as the Yale Coli Genetic Stock Collection (New Haven, Conn. USA).Strains developed from these strains are described in the Examples.

Bacterial growth culture media and associated materials and conditions,are as follows:

Fed-batch medium contained (per liter): 10 g tryptone, 5 g yeastextract, 1.5 g NaCl, 2 g Na₂HPO₄.7 H₂O, 1 g KH₂PO₄, and glucose asindicated

AM2 medium contained (per liter): 2.87 g K₂HPO₄, 1.50 g KH₂PO₄, 3.13 g(NH₄)₂SO₄, 0.15 g KCl, 1.5 mM MgSO₄, 0.1M K⁺ MOPS pH 7.2, 30 g glucose,and 1 ml trace Mineral Stock prepared as described in Martinez et al.Biotechnol Lett 29:397-404 (2007)

AM2 Medium used in Fermenters for Initial Batch Medium (for Example 11)

K₂HPO₄ 2.87 g/L KH₂PO₄ 1.50 g/L (NH₄)₂SO₄ 3.13 g/L KCl 0.15 g/L

Glucose 6.0 g/L MgSO₄ 0.18 g/L AM2 Trace Metals 1.0 ml/L Stock SolutionCalcium Pantothenate 0.005 g/L Ampicillin 0.1 g/L Kanamycin 0.02 g/LChloramphenicol 0.02 g/L

Trace Metals Stock Solution for AM2 medium used in Fermenters

Concentrated HCl 10.0 ml/L FeCl₃•6H₂O 2.4 g/L CoCl₂•6H₂O 0.17 g/LCuCl₂•2H₂O 0.15 g/L ZnCl₂ 0.3 g/L Na₂MoO₄•2H₂O 0.3 g/L H₃BO₃ 0.07 g/LMnCl₂•4H₂O 0.5 g/L

Concentration of glucose in glucose feed for AM2 vessels: 200 g/Lglucose

Rich Medium used in Fermenters Initial Batch Medium (for Example 11)

Tryptone 10 g/L Yeast Extract 5 g/L Glucose 4 g/L Na₂HPO₄•7H₂O 2 g/LKH₂PO₄ 1 g/L MgSO₄ 2 g/L Ampicillin 0.1 g/L Kanamycin 0.02 g/LChloramphenicol 0.02 g/L

Feed Formulation for additional glucose feed for rich media

Glucose 200 g/L (NH₄)₂SO₄ 30 g/L KH₂PO₄ 7.5 g/L Citric Acid 3 g/L MgSO₄2.93 g/L FeSO₄•7H₂O 0.05 g/L

SM3 minimal medium for E. coli (Final phosphate concentration=27.5 mM;Final N concentration=47.4 mM NH₄ ¹).

Components per liter: 700 mL, DI water, 100 mL 10× SM3 Salts, 2 ml 1MMgSO₄, 1 ml 1000× Trace Mineral Stock, 60 mL 500 g/L glucose, 100 mL 0.1M MOPS (pH 7.4), 0.1 mL, of 1 M CaCl₂, Q.S. with DI water to 1000 mL,and 0.2 μm filter sterilize.

Preparation of Stock Solutions:

To make 10× SM3 Salts (1 L): 800 mL DI water, 28.7 g K₂HPO₄, 15 gKH₂PO₄, 31.3 g (NH₄)₂SO₄, 1.5 g KCl, 0.5 g Citric Acid (anhydrous), andQ.S. with DI water to 1000 mL.

To make 1000× Trace Mineral Stock (1 L): save in 50-ml portions at roomtemp

Per liter in 0.12M HCl (dilute 10 ml cone HCl into 1 liter water): 2.4 gFeCl₃.6H₂O, 0.17 g CoCl₂.6H₂O, 0.15 g CuCl₂.2H₂O, 0.3 g ZnCl₂, 0.3 gNaMoO₄.2H₂O (Molybdic acid, disodium salt, dihydrate), 0.07 g H₃BO₃, and0.5 g MnCl₂.4H₂O.

To make 1M MOPS:209.3 g MOPS, dissolve in 700 ml water. Take 70-mlportions and adjust to desired pH with 50% KOH, adjust to 100 mL finalvolume, and 0.2 μm filter sterilize.

To make 1M MgSO):120.37 g dissolved in 1000 mL water.

To make 500 g/L (50%) glucose stock solution: 900 mL DI water,500 gglucose, and Q.S. to 1000 mL

Additional Growth Media Formulations are summarized as:

Concen- Concen- Concen- tration in tration in tration in Ingredient FM3FM4 FM5 1 K₂HPO₄ 2.63 g/L 13.4 g/L 2.63 g/L 2 KH₂PO₄ 1.38 g/L 3 g/L 1.38g/L 3 (NH₄)₂SO₄ 13.88 g/L 3 g/L 3 g/L 4 NaCl — 0.5 g/L — 5 CitricAcid•H₂O 2.19 g/L 1.1 g/L 2.19 g/L 6 Yeast Extract 1.25 g/L 1 g/L 1 g/L1 Antifoam 204 0.1 ml/L 0.1 ml/L 0.1 ml/L 8 Glucose 30 g/L 30 g/L 30 g/L9 MgSO₄•7H₂O 0.82 g/L 0.48 g/L 0.82 g/L 10 FM10 Trace Metals 1.5 ml/L 2ml/L 2 ml/L Stock Solution 11 Kanamycin 35 mg/L 35 mg/L 35 mg/L 12Chloramphenicol 20 mg/L 20 mg/L 20 mg/L

FM10: Trace Metals Stock Solution Formulation:

Ingredient Concentration Concentrated HCl 10.0 ml/L CaCl₂•2H₂O 49 g/LFeCl₃•6H₂O 9.7 g/L CoCl₂•6H₂0 0.4 g/L CuCl₂•2H₂O 2.7 g/L ZnCl₂ 0.2 g/LNa₂MoO₄•2H₂O 0.24 g/L H₃BO₃ 0.07 g/L MnCl₂•4H₂O 0.36 g/L

To make 1 L M9 minimal media:

M9 minimal media was made by combining 5× M9 salts, 1M MgSO₄, 20%glucose, 1M CaCl₂and sterile deionized water. The 5× M9 salts are madeby dissolving the following salts in deionized water to a final volumeof 1 L: 64 g Na₂HPO₄ 7H₂O, 15 g KH₂PO₄, 2.5 g NaCl, 5.0 g NH₄Cl. Thesalt solution was divided into 200 mL aliquots and sterilized byautoclaving for 15 minutes at 15 psi on the liquid cycle. A 1M solutionof MgSO₄ and 1M CaCl₂ were made separately, then sterilized byautoclaving. The glucose was filter sterilized by passing it though a0.22 μm filter. All of the components are combined as follows to make 1L of M9: 750 mL sterile water, 200 mL 5× M9 salts, 2 mL of 1M MgSO₄, 20mL 20% glucose, CaCl₂, Q.S. to a final volume of 1 L.

To make EZ Rich Media:

All media components were obtained from TEKnova (Hollister Calif. USA)and combined in the following volumes. 100 mL 10× MOPS mixture, 10 mL0.132M K₂ HPO₄, 100 mL 10× ACGU, 200 mL 5× Supplement EZ, 10 mL 20%glucose, 580 mL sterile water.

Subsection Gel Preparation, DNA Separation, Extraction, Ligation, andTransformation Methods:

Molecular biology grade agarose (RPI Corp, Mt. Prospect, Ill., USA) isadded to 1× TAE to make a 1% Agarose in TAE. To obtain 50× TAE add thefollowing to 900 ml distilled H₂O : 242 g Tris base (RPI Corp, Mt.Prospect, Ill., USA), 57.1 ml Glacial Acetic Acid (Sigma-Aldrich, St.Louis, Mo., USA), 18.6 g EDTA (Fisher Scientific, Pittsburgh, Pa. USA),and adjust volume to 1 L with additional distilled water. To obtain 1×TAE, add 20 mL of 50× TAE to 980 mL of distilled water. The agarose-TAEsolution is then heated until boiling occurred and the agarose is fullydissolved. The solution is allowed to cool to 50° C. before 10 mg/mLethidium bromide (Acros Organics, Morris Plains, N.J., USA) is added ata concentration of 5 ul per 100mL of 1% agarose solution. Once theethidium bromide is added, the solution is briefly mixed and poured intoa gel casting tray with the appropriate number of combs (Idea ScientificCo., Minneapolis, Minn., USA) per sample analysis. DNA samples are thenmixed accordingly with 5×TAE loading buffer. 5×TAE loading bufferconsists of 5× TAE (diluted from 50× TAE as described herein), 20%glycerol (Acros Organics, Morris Plains, N.J., USA), 0.125% BromophenolBlue (Alfa Aesar, Ward Hill, Mass., USA), and adjust volume to 50 mLwith distilled water. Loaded gels are then run in gel rigs (IdeaScientific Co., Minneapolis, Minn., USA) filled with 1× TAE at aconstant voltage of 125 volts for 25-30 minutes. At this point, the gelsare removed from the gel boxes with voltage and visualized under a UVtransilluminator (FOTODYNE Inc., Hartland, Wis., USA),

The DNA isolated through gel extraction is then extracted using theQIAquick Gel Extraction Kit following manufacturer's instructions(Qiagen (Valencia Calif. USA)). Similar methods are known to thoseskilled in the art.

The thus-extracted DNA then may be ligated into pSMART (Lucigen Corp,Middleton, Wis., USA), StrataClone (Stratagene, La Jolla, Calif., USA)or pCR2.1-TOPO TA (Invitrogen Corp, Carlsbad, Calif., USA) according tomanufacturer's instructions. These methods are described in the nextsubsection of Common Methods.

Ligation Methods:

For ligations into pSMART vectors:

Gel extracted DNA is blunted using PCRTerminator (Lucigen Corp,Middleton, Wis., USA) according to manufacturer's instructions. Then 500ng of DNA is added to 2.5 uL 4× CloneSmart vector premix, 1 ulCloneSmart DNA ligase (Lucigen Corp, Middleton, Wis., USA) and distilledwater is added for a total volume of 10 ul. The reaction is then allowedto sit at room temperature for 30 minutes and then heat inactivated at70° C. for 15 minutes and then placed on ice. E. cloni 10G ChemicallyCompetent cells (Lucigen Corp, Middleton, Wis., USA) are thawed for 20minutes on ice. 40 ul of chemically competent cells are placed into amicrocentrifuge tube and 1 ul of heat inactivated CloneSmart Ligation isadded to the tube. The whole reaction is stirred briefly with a pipettetip. The ligation and cells are incubated on ice for 30 minutes and thenthe cells are heat shocked for 45 seconds at 42° C. and then put backonto ice for 2 minutes. 960 ul of room temperature Recovery media(Lucigen Corp, Middleton, Wis., USA) and places into microcentrifugetubes. Shake tubes at 250 rpm for hour at 37° C. Plate 100 ul oftransformed cells on Luria Broth plates (RPI Corp, Mt. Prospect, Ill.,USA) plus appropriate antibiotics depending on the pSMART vector used.Incubate plates overnight at 37° C.

For Ligations into StrataClone:

Gel extracted DNA is blunted using PCRTerminator (Lucigen Corp,Middleton, Wis., USA) according to manufacturer's instructions. Then 2ul of DNA is added to 3 ul StrataClone Blunt Cloning buffer and 1 ulStrataClone Blunt vector nix amp/kan (Stratagene, La Jolla, Calif., USA)for a total of 6 ul. Mix the reaction by gently pipeting up at down andincubate the reaction at room temperature for 30 minutes then place ontoice. Thaw a tube of StrataClone chemically competent cells (Stratagene,La Jolla, Calif., USA) on ice for 20 minutes. Add 1 ul of the cloningreaction to the tube of chemically competent cells and gently mix with apipette tip and incubate on ice for 20 minutes. Heat shock thetransformation at 42° C. for 45 seconds then put on ice for 2 minutes,Add 250 ul pre-warmed Luria Broth (RPI Corp, Mt. Prospect, Ill., USA)and shake at 250 rpm for 37° C. for 2 hour. Plate 100 ul of thetransformation mixture onto Luria Broth plates (RPI Corp, Mt. Prospect,Ill., USA) plus appropriate antibiotics. Incubate plates overnight at37° C.

For Ligations into pCR2.1-TOPO TA:

Add 1 ul TOPO vector, 1 ul Salt Solution (Invitrogen Corp, Carlsbad,Calif., USA) and 3 ul gel extracted DNA into a microcentrifuge tube.Allow the tube to incubate at room temperature for 30 minutes then placethe reaction on ice. Thaw one tube of TOP1- F′ chemically competentcells (Invitrogen Corp, Carlsbad, Calif., USA) per reaction. Add 1 ul ofreaction mixture into the thawed TOP10F′ cells and mix gently byswirling the cells with a pipette tip and incubate on ice for 20minutes. Heat shock the transformation at 42° C. for 45 seconds then puton ice for 2 minutes. Add 250 ul pre-warmed SOC media (Invitrogen Corp,Carlsbad, Calif., USA) and shake at 250 rpm for 37° C. for 1 hour. Plate100 ul of the transformation mixture onto Luria Broth plates (RPI Corp,Mt. Prospect, Ill., USA) plus appropriate antibiotics. Incubate platesovernight at 37° C.

General Transformation and Related Culture Methodologies:

Chemically competent transformation protocols are carried out accordingto the manufacturer's instructions or according to the literaturecontained in Molecular Cloning (Sambrook and Russell, 2001). Generally,plasmid DNA or ligation products are chilled on ice for 5 to 30 min. Insolution with chemically competent cells. Chemically competent cells area widely used product in the field of biotechnology and are availablefrom multiple vendors, such as those indicated in this Subsection.Following the chilling period cells generally are heat-shocked for 30seconds at 42° C. without shaking, re-chilled and combined with 250microliters of rich media, such as S.O.C. Cells are then incubated at37° C. while shaking at 250 rpm for 1 hour. Finally, the cells arescreened for successful transformations by plating on media containingthe appropriate antibiotics.

Alternatively, selected cells may be transformed by electroporationmethods such as are known to those skilled in the art.

The choice of an E. coli host strain for plasmiod transformation isdetermined by considering factors such as plasmid stability, plasmidcompatibility, plasmid screening methods and protein expression. Strainbackgrounds can be changed by simply purifying plasmid DNA as describedherein and transforming the plasmid into a desired or otherwiseappropriate E. coli host strain such as determined by experimentalnecessities, such as any commonly used cloning strain (e.g., DH5α,Top10F′, E. cloni 10G, etc.).

Plasmid DNA was prepared using the commercial miniprep kit from Qiagen(Valencia, Calif. USA) according to manufacturer's instructions.

Subsection IIIa, 3-HP Preparation

A 3-HP stock solution was prepared as follows. A vial ofβ-propriolactone (Sigma-Aldrich, St. Louis, Mo., USA) was opened under afume hood and the entire bottle contents was transferred to a newcontainer sequentially using a 25 ml glass pipette. The vial was rinsedwith 50 mL of HPLC grade water and this rinse was poured into the newcontainer. Two additional rinses were performed and added to the newcontainer. Additional HPLC grade water was added to the new container toreach a ratio of 50 mL water per 5 mL propriolactone. The new containerwas capped tightly and allowed to remain in the fume hood at roomtemperature for 72 hours. After 72 hours the contents were transferredto centrifuge tubes and centrifuged for 10 minutes at 4,000 rpm. Thenthe solution was filtered to remove particulates and, as needed,concentrated by use of a rotary evaporator at room temperature. Assayfor concentration was conducted, and dilution to make a standardconcentration stock solution was made as needed.

Subsection IIIb, HPLC, GC/MS and Other Analytical Methods for 3-HPDetection (Analysis of Cultures for 3-HP Production)

For HPLC analysis of 3-HP, the Waters chromatography system (Milford,Mass.) consisted of the following: 600S Controller, 616 Pump, 717 PlusAutosampler, 486 Triable UV Detector, and an in-line mobile phaseDegasser. In addition, an Eppendorf external column heater is used andthe data are collected using an SRI (Torrance, Calif.) analog-to-digitalconverter linked to a standard desk top computer. Data are analyzedusing the SRI Peak Simple software. A Coregel 64H ion exclusion column(Transgenomic, Inc., San Jose, Calif.) is employed. The column resin isa sulfonated polystyrene divinyl benzene with a particle size of 10 μmand column dimensions are 300×7.8 mm. The mobile phase consisted ofsulfuric acid (Fisher Scientific, Pittsburgh, Pa. USA) diluted withdeionized (18 MΩcm) water to a concentration of 0.02 N and vacuumfiltered through a 0.2 μm nylon filter. The flow rate of the mobilephase is 0.6 mL/min. The UV detector is operated at a wavelength of 210nm and the column is heated to 60° C. The same equipment and method asdescribed herein is used for 3-HP analyses for relevant propheticexamples. A representative calibration curve using this HPLC method witha 3-HP standard (TCI America, Portland, Oreg.) is provided in FIG. 13.

The following method is used for GC-MS analysis of 3-HP. Solublemonomeric 3-HP is quantified using GC-MS after a single extraction ofthe fermentation media with ethyl acetate. Once the 3-HP has beenextracted into the ethyl acetate, the active hydrogens on the 3-HP arereplaced with trimethylsilyl groups usingN,O-Bis-(Trimethyisilyl)trifluoroacetamide to make the compound volatilefor GC analysis. A standard curve of known 3-HP concentrations isprepared at the beginning of the run and a known quantity ofketohexanoic acid (1 g/L) is added to both the standards and the samplesto act as an internal standard for Quantitation, with tropic acid as anadditional internal standard. The 3-HP content of individual samples isthen assayed by examining the ratio of the ketohexanoic acid ion(m/z=247) to the 3-HP ion (219) and compared to the standard curve. 3-HPis quantified using a 3HP standard curve at the beginning of the run andthe data are analyzed using HP Chemstation. The GC-MS system consists ofa Hewlett Packard model 5890 GC and Hewlett Packard model 5972 MS. Thecolumn is Supelco SPB-1 (60 m×0.32 mm×0.25 μm film thickness). Thecapillary coating is a non-polar methylsilicone. The carrier gas ishelium at a flow rate of 1 mL/min. The 3-HP as derivatized is separatedfrom other components in the ethyl acetate extract using either of twosimilar temperature regimes. In a first temperature gradient regime, thecolumn temperature starts with 40° C. for 1 minute, then is raised at arate of 10° C./minute to 235° C., and then is raised at a rate of 50°C./minute to 300° C. In a second temperature regime, which wasdemonstrated to process samples more quickly, the column temperaturestarts with 70° C. which is held for 1 min, followed by a ramp-up of 10°C./minute to 235° C. which is followed by a ramp-up of 50° C./minute to300° C. A representative calibration curve is provided in FIG. 22.

A bioassay for detection of 3-HP also was used in various examples. Thisdetermination of 3-HP concentration was carried out based on theactivity of the E. coli 3-HP dehydrogenase encoded by the ydfG gene (theYDFG protein). Reactions of 200-μl were carried out in 96-wellmicrotiter plates, and contained 100 mM Tris-HCl, pH 8.8, 2.5 mM MgCl₂,2.625 mM NADP⁺, 3 μg purified YDFG and 20 μl culture supernatant.Culture supernatants were prepared by centrifugation in a microtiage(14,000 rpm, 5 min) to remove cells. A standard curve of 3-HP(containing from 0.025 to 2 g/l) was used in parallel reactions toquantitate the amount of 3-HP in culture supernatants. Uninoculatedmedium was used as the reagent blank. Where necessary, the culturesupernatant was diluted in medium to obtain a solution with 3-HPconcentrations within that of the standard curve.

The reactions were incubated at 37° C. for 1 hr, and 20 μl of colordeveloper containing 1.43 mM nitroblue tetrazolium, 0.143 phenazinemethosulfate, and 2.4% bovine serum albumin were added to each reaction.Color development was allowed to proceed at 37° C. for an additional hr,and the absorbance at 580 nm was measured. 3-HP concentration in theculture supernatants was quantitated by comparison with the valuesobtained from the standard curve generated on the same microtiter plate.The results obtained with the enzymatic assay were verified to matchthose obtained by one of the analytical methods described above. FIG. 23provides a representative standard curve.

Subsection IV. Minimum Inhibitory Concentration Evaluation (MIC)Protocols

For MIC evaluations, the final results are expressed in chemical agentconcentrations determined by analysis of the stock solution by HPLC(i.e., see Subsection IIIb).

E. coli Aerobic

The minimum inhibitory concentration (MIC) was determined aerobically ina 96 well-plate format. Plates were setup such that each individualwell, when brought to a final volume of 100 uL following inoculation,had the following component levels (corresponding to standard M9 media):47.7 mM Na₂HPO₄, 22 mM KH₂PO₄.8.6 mM NaCl, 18.7 mM NH₄Cl, 2 mM MgSO₄,0.1 mM CaCl₂, and 0.4% glucose. Media supplements were added accordingto levels reported in the Supplements Table, where specified. Overnightcultures of strains were grown in triplicate in 5 mL LB (with antibioticwhere appropriate). A 1% (v/v) inoculum was introduced into a 5 mlculture of M9 minimal media. After the cells reached mid-exponentialphase, the culture was diluted to an OD₆₀₀ of about 0.200 (i.e.,0.195-0.205. The cells were further diluted 1:50 and a 10 μL aliquot wasused to inoculate each well of a 96 well plate (˜10⁴ cells per well) tototal volume of 100 uL. The plate was arranged to measure the growth ofvariable strains or growth conditions in increasing 3-HP concentrations,0 to 60 g/L, in 5 g/L increments. Plates were incubated for 24 hours at37 C. The minimum inhibitory 3-HP concentration and maximum 3-HPconcentration corresponding to visible cell growth (OD˜0.1) was recordedafter 24 hours. For cases when MIC >60 g/L, assessments were performedin plates with extended 3-HP concentrations (0-100 g/L, in 5 g/Lincrements).

E. Coli Anaerobic

The minimum inhibitory concentration (MIC) was determined anaerobicallyin a 96 well-plate format. Plates were setup such that each individualwell, when brought to a final volume of 100 uL following inoculation,had the following component levels (corresponding to standard M9 media):47.7 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.6 mM NaCl, 18.7 mM NH₄Cl, 2 mM. MgSO₄,0.1 mM CaCl₂, and 0.4% glucose. Media supplements were added accordingto levels reported in the Supplements Table, where specified. Overnightcultures of strains were grown in triplicate in 5 mL LB (with antibioticwhere appropriate). A 1% (v/v) inoculum was introduced into a 5 mlculture of M9 minimal media. After the cells reached mid-exponentialphase, the culture was diluted to an OD₆₀₀ of about 0.200 (i.e.,0.195-0.205. The cells were further diluted 1:50 and a 10 μL aliquot wasused to inoculate each well of a 96 well plate (˜10⁴ cells per well) tototal volume of 100 uL. The plate was arranged to measure the growth ofvariable strains or growth conditions in increasing 3-HP concentrations,0 to 60 g/L, in 5 g/L increments. Plates were sealed in biobag anaerobicchambers that contained gas generators for anaerobic conditions andincubated for 24 hours at 37 C. The minimum inhibitory 3-HPconcentration and maximum 3-HP concentration corresponding to visiblecell growth (OD˜0.1) was recorded after 24 hours. For cases when MIC >60g/L, assessments were performed in plates with extended 3-HPconcentrations (0-100 g/L, in 5 g/L increments).

B. Subtilis Aerobic

The minimum inhibitory concentration (MIC) was determined aerobically ina 96 well-plate format Plates were setup such that each individual well,when brought to a final volume of 100 uL following inoculation, had thefollowing component levels (corresponding to standard M9media+supplemental glutamate): 47.7 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.6 mMNaCl, 18.7 mM NH₄Cl, 2 mM MgSO₄, 0.1 mM CaCl₂, 10 mM glutamate and 0.4%glucose. Media supplements were added according to levels reported inthe Supplements Table where specified. Overnight cultures of strainswere grown in triplicate in 5 mL LB (with antibiotic where appropriate).A 1% (v/v) inoculum was introduced into a 5 ml culture of M9 minimalmedia+glutamate. After the cells reached mid-exponential phase, theculture was diluted to an OD ₆₀₀ of about 0.200 (i.e., 0.195-0.205. Thecells were further diluted 1:50 and a 10 μL aliquot was used toinoculate each well of a 96 well plate (˜10⁴ cells per well) to totalvolume of 100 uL. The plate was arranged to measure the growth ofvariable strains or growth conditions in increasing 3-HP concentrations,0 to 60 g/L, in 5 g/L increments. Plates were incubated for 24 hours at37 C. The minimum inhibitory 3-HP concentration and maximum 3-HPconcentration corresponding to visible cell growth (OD˜0.1) was recordedafter 24 hours. For cases when MIC >60 g/L, assessments were performedin plates with extended 3-HP concentrations (0-100 g/L, in 5 g/Lincrements),

C. Necator (R. Eutropha) Aerobic

The minimum inhibitory concentration (MIC) was determined aerobically ina 96-plate format. Plates were setup such that each individual well,when brought to a final volume of 100 uL following inoculation, had thefollowing component levels (corresponding to FGN media): 21.5 mM K₂HPO₄,8.5 mM KH₂PO₄, 18 mM NH₄Cl, 12 mM NaCl, 7.3 uM ZnCl, 0.15 uM MnCl₂, 4.85uM H₃BO₃, 0.21 uM CoCl₂, 0.41 uM CuCl₂, 0.50 uM NiCl₂, 0.12 uM Na₂MoO₄.0.19 uM CrCl₃, 0.06 mM CaCl₂, 0.5 mM MgSO₄, 0.06 mM FeSO₄, 0.2%glycerol, 0.2% fructose. Media supplements were added according tolevels reported in Supplements Table, where specified. Overnightcultures of strains were grown in triplicate in 5 mL LB (with antibioticwhere appropriate). A 1% (v/v) inoculum was introduced into a 5 mlculture of FGN media. After the cells reached mid-exponential phase, theculture was diluted to an OD₆₀₀ of about 0.200 (i.e., 0.195-0.205. Thecells were further diluted 1:50 and a 10 μL aliquot was used toinoculate each well of a 96 well plate (˜10⁴ cells per well) to totalvolume of 100 uL. The plate was arranged to measure the growth ofvariable strains or growth conditions in increasing 3-HP concentrations,0 to 60 g/L, in 5 g/L increments. Plates were incubated for 24 hours at30 C. The mini mm inhibitory 3-HP concentration and maximum 3-HPconcentration corresponding to visible cell growth (OD˜0.1) was recordedafter 24 hours. For cases when MIC >60 g/L, assessments were performedin plates with extended 3-HP concentrations (0-100 g/L, in 5 g/Lincrements).

The embodiments, variations, sequences, and figures described hereinshould provide an indication of the utility and versatility of thepresent invention. Other embodiments that do not provide all of thefeatures and advantages set forth herein may also be utilized, withoutdeparting from the spirit and scope of the present invention. Suchmodifications and variations are considered to be within the scope ofthe invention.

1. (canceled)
 2. A method for producing a product having malonyl-CoA asa substrate microbial production pathway of the product, the methodcomprising combining a carbon source and a microorganism cell culture toproduce said product, wherein a) said microorganism is geneticallymodified for reduced enzymatic activity of a thioesterase, and b) saidmicroorganism is genetically modified for overexpression of accA, accB,accC, accD, or any combination thereof.
 3. A method for producing aproduct having malonyl-CoA as a substrate in a microbial productionpathway of the product, the method comprising combining a carbon sourceand a microorganism cell culture to produce said product, wherein a)said microorganism is genetically modified for reduced enzymaticactivity of a phosphotransferase (PTS), and b) said microorganism isgenetically modified for overexpression of accA, accB, accC, accD, orany combination thereof.
 4. A method for producing a product havingmalonyl-CoA as a substrate microbial production pathway of the product,the method comprising combining a carbon source and a microorganism cellculture to produce said product, wherein a) said microorganism isgenetically modified for increased enzymatic activity of a pyruvatedehydrogenase (Pdh), and b) said microorganism is genetically modifiedfor overexpression of accA, accB, accC, accD, or any combinationthereof.
 5. The method of claim 2, wherein said microorganismadditionally is genetically modified for reduced enzymatic activity of aphosphotransferase (PTS), or for increased enzymatic activity of apyruvate dehydrogenase (Pdh), or both.
 6. The method of claim 3, whereinsaid microorganism additionally is genetically modified for increasedenzymatic activity of a pyruvate dehydrogenase (Pdh).